Compounds for enzyme inhibition

ABSTRACT

Peptide-based compounds including heteroatom-containing, three-membered rings efficiently and selectively inhibit specific activities of N-terminal nucleophile (Ntn) hydrolases associated with the proteasome. The peptide-based compounds include an epoxide or aziridine, and functionalization at the N-terminus. Among other therapeutic utilities, the peptide-based compounds are expected to display anti-inflammatory properties and inhibition of cell proliferation. Oral administration of these peptide-based proteasome inhibitors is possible due to their bioavailability profiles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.13/736,605, filed Jan. 8, 2013, which is a continuation of U.S.application Ser. No. 12/084,838, filed Aug. 7, 2008, which is a NationalStage application under 35 U.S.C. §371 of International Application No.PCT/US2006/043503, having an International Filing Date of Nov. 9, 2006,which claims the benefit of U.S. Provisional Application Ser. No.60/842,582, filed Sep. 5, 2006, and U.S. Provisional Application Ser.No. 60/736,118, filed Nov. 9, 2005. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I presentation,apoptosis, cell division, and NF-κB activation.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings thatplays important roles in cell growth regulation, majorhistocompatibility complex class I presentation, apoptosis, antigenprocessing, NF-κB activation, and transduction of pro-inflammatorysignals. In yeast and other eukaryotes, 7 different α subunits form theouter rings and 7 different β subunits comprise the inner rings. The αsubunits serve as binding sites for the 19S (PA700) and 11S (PA28)regulatory complexes, as well as a physical barrier for the innerproteolytic chamber formed by the two β subunit rings. Thus, in vivo,the proteasome is believed to exist as a 26S particle (“the 26Sproteasome”). In vivo experiments have shown that inhibition of the 20Sform of the proteasome can be readily correlated to inhibition of 26Sproteasome. Cleavage of amino-terminal prosequences of β subunits duringparticle formation expose amino-terminal threonine residues, which serveas the catalytic nucleophiles. The subunits responsible for catalyticactivity in proteasome thus possess an amino terminal nucleophilicresidue, and these subunits belong to the family of N-terminalnucleophile (Ntn) hydrolases (where the nucleophilic N-terminal residueis, for example, Cys, Ser, Thr, and other nucleophilic moieties). Thisfamily includes, for example, penicillin G acylase (PGA), penicillin Vacylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three γ-interferon-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, X, Y and Z respectively, thus altering the catalyticactivities of the proteasome. Through the use of different peptidesubstrates, three major proteolytic activities have been defined for theeukaryote 20S proteasome: chymotrypsin-like activity (CT-L), whichcleaves after large hydrophobic residues; trypsin-like activity (T-L),which cleaves after basic residues; and peptidylglutamyl peptidehydrolyzing activity (PGPH), which cleaves after acidic residues. Twoadditional less characterized activities have also been ascribed to theproteasome: BrAAP activity, which cleaves after branched-chain aminoacids; and SNAAP activity, which cleaves after small neutral aminoacids. The major proteasome proteolytic activities appear to becontributed by different catalytic sites, since inhibitors, pointmutations in β subunits and the exchange of γ interferon-inducing βsubunits alter these activities to various degrees.

In recent years, the proteasome has become an appealing target fortherapeutic intervention in cancer, immune and auto-immune disorders,inflammation, ischemic conditions, neurodegenerative disorders and otherdiseases. To date, the only FDA-approved proteasome inhibitor isbortezomib (VELCADE™), however, several other proteasome inhibitors arecurrently being evaluated in clinical trials. Thus far, all thesetherapeutic proteasome inhibitors currently are administered via IV.Clinical application of proteasome inhibitors in the treatment ofhematologic malignancies such as myeloma and lymphoma is restricted inpart by the necessity of frequent IV administrations and would beimproved by oral (PO) administration. However, due to the peptide natureof these molecules, systemic exposure following PO administration ofthese inhibitors is limited by several factors including gastric pH,gastric and intestinal peptidases, efflux pumps, biliary excretion andintestinal and hepatic metabolic activities.

Methods used to overcome the ability of peptides to be enzymaticallydegraded and to improve absorption into the blood stream from thedigestive tract have included making analogs which are less peptide-likein structure and which are reduced in size. Such methods are deemed tobe successful when the peptide analog achieves satisfactory blood levelsafter oral administration, or in the case of proteasome inhibitors, whenthe proteasome activity in blood is satisfactorily reduced.

The above mentioned techniques have been applied to preparing analogs ofthe peptide epoxyketone proteasome inhibitors, thereby rendering themorally bioavailable.

SUMMARY OF THE INVENTION

The invention relates to classes of molecules known as peptideα′,β′-epoxides and peptide α′,β′-aziridines. The parent molecules areunderstood to bind efficiently, irreversibly and selectively toN-terminal nucleophile (Ntn) hydrolases, and can specifically inhibitparticular activities of enzymes having multiple catalytic activity.

Once thought merely to dispose of denatured and misfolded proteins, theproteasome is now recognized as constituting proteolytic machinery thatregulates the levels of diverse intracellular proteins through theirdegradation in a signal-dependent manner. Hence, there is great interestin identifying reagents that can specifically perturb the activities ofthe proteasome and other Ntn hydrolases and thereby be used as probes tostudy the role of these enzymes in biological processes. Compounds thattarget the Ntn hydrolases are herein described, synthesized, andinvestigated. Peptide epoxides and peptide aziridines that can potently,selectively, and irreversibly inhibit particular proteasome activitiesare disclosed and claimed.

Unlike several other peptide-based inhibitors, the peptide epoxides andpeptide aziridines described herein are not expected to substantiallyinhibit non-proteasomal proteases such as trypsin, chymotrypsin,cathepsin B, papain, and calpain at concentrations up to 50 μM. Athigher concentrations, inhibition may be observed, but would be expectedto be competitive and not irreversible, if the inhibitor merely competeswith the substrate. The novel peptide epoxides and peptide aziridinesare also expected to inhibit NF-κB activation and to stabilize p53levels in cell culture. Moreover, these compounds would be expected tohave anti-inflammatory activity. Thus, these compounds can be uniquemolecular probes, which have the versatility to explore Ntn enzymefunction in normal biological and pathological processes.

In one aspect, the invention provides inhibitors comprising aheteroatom-containing three-membered ring. These inhibitors can inhibitcatalytic activity of N-terminal nucleophile hydrolase enzymes (forexample, the 20S proteasome, or the 26S proteasome) when said inhibitoris present at concentrations below about 50 μM. Regarding the 20Sproteasome, particular hydrolase inhibitors inhibit chymotrypsin-likeactivity of the 20S proteasome when the inhibitor is present atconcentrations below about 5 μM, and does not inhibit trypsin-likeactivity or PGPH activity of the 20S proteasome when present atconcentrations below about 5 μM. The hydrolase inhibitor may be, forexample, a peptide α′,β′-epoxy ketone or α′,β′-aziridine ketone, and thepeptide may be a tetrapeptide. The peptide may include branched orunbranched side chains such as hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, C₁₋₆aralkyl, C₁₋₆alkylamide, C₁₋₆alkylamine,C₁₋₆-carboxylic acid, C₁₋₆-carboxyl ester, C₁₋₆alkylthiol, orC₁₋₆alkylthioether, for example isobutyl, 1-naphthyl, phenylmethyl, and2-phenylethyl. The α′-carbon of the α′,β′-epoxy ketone orα′,β′-aziridine ketone may be a chiral carbon atom, such as an (R) or βconfigured carbon, as these are defined herein.

In another aspect, the invention provides pharmaceutical compositions,including a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of the hydrolase inhibitor, which ameliorates theeffects of neurodegenerative disease (such as Alzheimer's disease),muscle-wasting diseases, cancer, chronic infectious diseases, fever,muscle disuse, denervation, nerve injury, fasting, and immune-relatedconditions, among others.

In another aspect, the invention provides compounds and pharmaceuticalcompositions that are orally bioavailable.

In another aspect, the invention provides anti-inflammatorycompositions.

In another aspect, the invention provides methods for the following:inhibiting or reducing HIV infection in a subject; affecting the levelof viral gene expression in a subject; altering the variety of antigenicpeptides produced by the proteasome in an organism; determining whethera cellular, developmental, or physiological process or output in anorganism is regulated by the proteolytic activity of a particular Ntnhydrolase; treating Alzheimer's disease in a subject; reducing the rateof muscle protein degradation in a cell; reducing the rate ofintracellular protein degradation in a cell; reducing the rate of p53protein degradation in a cell; inhibiting the growth of p53-relatedcancers in a subject; inhibiting antigen presentation in a cell;suppressing the immune system of a subject; inhibiting IκB-α degradationin an organism; reducing the content of NF-κB in a cell, muscle, organor subject; affecting cyclin-dependent eukaryotic cell cycles; treatingproliferative disease in a subject; affecting proteasome-dependentregulation of oncoproteins in a cell; treating cancer growth in asubject; treating p53-related apoptosis in a subject; and screeningproteins processed by N-terminal nucleophile hydrolases in a cell. Eachof these methods involves administering or contacting an effectiveamount of a composition comprising the hydrolase inhibitors disclosedherein, to a subject, a cell, a tissue, an organ, or an organism.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves compounds useful as enzyme inhibitors. Thesecompounds are generally useful to inhibit enzymes having a nucleophilicgroup at the N-terminus. For example, activities of enzymes or enzymesubunits having N-terminal amino acids with nucleophiles in their sidechains, such as threonine, serine, or cysteine can be successfullyinhibited by the enzyme inhibitors described herein. Activities ofenzymes or enzyme subunits having non-amino acid nucleophilic groups attheir N-termini, such as, for example, protecting groups orcarbohydrates, can also be successfully inhibited by the enzymeinhibitors described herein.

While not bound by any particular theory of operation, it is believedthat such N-terminal nucleophiles of Ntn form covalent adducts with theepoxide functional group of the enzyme inhibitors described herein. Forexample, in the β5/Pre2 subunit of 20S proteasome, the N-terminalthreonine is believed to irreversibly form a morpholino or piperazinoadduct upon reaction with a peptide epoxide or aziridine such as thosedescribed below. Such adduct formation would involve ring-openingcleavage of the epoxide or aziridine.

In embodiments including such groups bonded to α′ carbons, thestereochemistry of the α′-carbon (that carbon forming a part of theepoxide or aziridine ring) can be (R) or (S). The invention is based, inpart, on the structure-function information disclosed herein, whichsuggests the following preferred stereochemical relationships. Note thata preferred compound may have a number of stereocenters having theindicated up-down (or β-α, where β as drawn herein is above the plane ofthe page) or (R)—(S) relationship (that is, it is not required thatevery stereocenter in the compound conform to the preferences stated).In some preferred embodiments, the stereochemistry of the α′ carbon is(R), that is, the X atom is β, or above the plane of the molecule.

Regarding the stereochemistry, the Cahn-Ingold-Prelog rules fordetermining absolute stereochemistry are followed. These rules aredescribed, for example, in Organic Chemistry, Fox and Whitesell; Jonesand Bartlett Publishers, Boston, Mass. (1994); Section 5-6, pp 177-178,which section is hereby incorporated by reference. Peptides can have arepeating backbone structure with side chains extending from thebackbone units. Generally, each backbone unit has a side chainassociated with it, although in some cases, the side chain is a hydrogenatom. In other embodiments, not every backbone unit has an associatedside chain. Peptides useful in peptide epoxides or peptide aziridineshave two or more backbone units. In some embodiments useful forinhibiting chymotrypsin-like (CT-L) activity of the proteasome, betweentwo and four backbone units are present, and in some preferredembodiments for CT-L inhibition, three backbone units are present.

The side chains extending from the backbone units can include naturalaliphatic or aromatic amino acid side chains, such as hydrogen(glycine), methyl (alanine), isopropyl (valine), sec-butyl (isoleucine),isobutyl (leucine), phenylmethyl (phenylalanine), and the side chainconstituting the amino acid proline. The side chains can also be otherbranched or unbranched aliphatic or aromatic groups such as ethyl,n-propyl, n-butyl, t-butyl, and aryl substituted derivatives such as1-phenylethyl, 2-phenylethyl, (1-naphthyl)methyl, (2-naphthyl)methyl,1-(1-naphthyl)ethyl, 1-(2-naphthyl)ethyl, 2-(1-naphthyl)ethyl,2-(2-naphthyl)ethyl, and similar compounds. The aryl groups can befurther substituted with branched or unbranched C₁₋₆alkyl groups, orsubstituted alkyl groups, acetyl and the like, or further aryl groups,or substituted aryl groups, such as benzoyl and the like. Heteroaryl andheterocyclyl groups can also be used as side chain substituents.Heteroaryl groups include nitrogen-, oxygen-, and sulfur-containing arylgroups such as thienyl, benzothienyl, naphthothienyl, thianthrenyl,furyl, pyranyl, isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl,pyrazolyl, pyridyl, pyrazinyl, indolyl, purinyl, quinolyl, and the like.Heterocyclyl groups include tetrahydrofuran, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

In some embodiments, polar or charged residues can be introduced intothe peptide epoxides or peptide aziridines. For example, naturallyoccurring amino acids such as hydroxy-containing (Thr, Tyr, Ser) orsulfur-containing (Met, Cys) can be introduced, as well as non-essentialamino acids, for example, taurine, carnitine, citrulline, cystine,ornithine, norleucine and others. Non-naturally occurring side chainsubstituents with charged or polar moieties can also be included, suchas, for example, C₁₋₆alkyl chains or C₆₋₁₂aryl groups with one or morehydroxy, short chain alkoxy, sulfide, thio, carboxyl, ester, phospho,amido or amino groups, or such substituents substituted with one or morehalogen atoms. In some preferred embodiments, there is at least one arylgroup present in a side chain of the peptide moiety.

In some embodiments, the backbone units are amide units[—NH—CHR—C(═O)—], in which R is the side chain. Such a designation doesnot exclude the naturally occurring amino acid proline, or othernon-naturally occurring cyclic secondary amino acids, which will berecognized by those of skill in the art.

In other embodiments, the backbone units are N-alkylated amide units(for example, N-methyl and the like), olefinic analogs (in which one ormore amide bonds are replaced by olefinic bonds), tetrazole analogs (inwhich a tetrazole ring imposes a cis-configuration on the backbone), orcombinations of such backbone linkages. In still other embodiments, theamino acid α-carbon is modified by α-alkyl substitution, for example,aminoisobutyric acid. In some further embodiments, side chains arelocally modified, for example, by Δ^(E) or Δ^(Z) dehydro modification,in which a double bond is present between the α and β atoms of the sidechain, or for example by Δ^(E) or Δ^(Z) cyclopropyl modification, inwhich a cyclopropyl group is present between the α and β atoms of theside chain. In still further embodiments employing amino acid groups,D-amino acids can be used. Further embodiments can include sidechain-to-backbone cyclization, disulfide bond formation, lactamformation, azo linkage, and other modifications discussed in “Peptidesand Mimics, Design of Conformationally Constrained” by Hruby and Boteju,in “Molecular Biology and Biotechnology: A Comprehensive DeskReference”, ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664,which is hereby incorporated by reference.

One aspect of the invention relates to compounds having a structure offormula (I) or a pharmaceutically acceptable salt thereof:

wherein

L is selected from C═O, C═S, and SO₂, preferably C═O;

X is selected from O, S, NH, and N—C₁₋₆alkyl;

Z is absent, C₁₋₆alkyl, or C₁₋₆alkoxy, preferably absent;

R¹, R², and R³ are each independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,C₁₋₆aralkyl, heteroaryl, heterocyclyl, C₁₋₆heterocycloalkyl,C₁₋₆heteroaralkyl, carbocyclyl, and C₁₋₆-carbocyclolalkyl;

R⁴ is selected from hydrogen, C₁₋₆aralkyl, and C₁₋₆alkyl;

R⁵ is heteroaryl; and

R⁶ and R⁷ are independently selected from hydrogen, C₁₋₆alkyl, andC₁₋₆aralkyl.

In certain embodiments, R¹, R², and R³ are independently selected fromhydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, C₁₋₆heteroaralkyl, and C₁₋₆-carbocyclolalkyl. Incertain embodiments, any of R¹, R², and R³ are independently C₁₋₆alkylselected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, andisobutyl. In certain embodiments, any of R¹, R², and R³ areindependently C₁₋₆hydroxyalkyl. In certain preferred such embodiments,any of R¹, R², and R³ are independently selected from hydroxymethyl andhydroxyethyl, preferably hydroxymethyl. In certain embodiments, any ofR¹, R², and R³ are independently C₁₋₆alkoxyalkyl. In certain suchembodiments, any of R¹, R², and R³ are independently selected frommethoxymethyl and methoxyethyl, preferably methoxymethyl. In certainembodiments, any of R¹, R², and R³ are independently C₁₋₆heteroaralkyl.In certain such embodiments, any of R¹, R², and R³ are independentlyselected from imidazolylmethyl, pyrazolylmethyl, and thiazolylmethyl,and pyridylmethyl, preferably imidazol-4-ylmethyl, thiazol-4-ylmethyl,2-pyridylmethyl, 3-pyridylmethyl, or 4-pyridylmethyl. In certainembodiments, any of R¹, R², and R³ are independently C₁₋₆aralkyl. Incertain such embodiments, any of R¹, R², and R³ are independentlyselected from phenylmethyl (benzyl) and phenylethyl, preferablyphenylmethyl. In certain embodiments, any of R¹, R², and R³ areindependently C₁₋₆carbocycloalkyl. In certain such embodiments R¹ iscyclohexylmethyl. In certain embodiments R¹, R², and R³ are alldifferent. In certain embodiments, any two of R¹, R², and R³ are thesame. In certain embodiments, R¹, R², and R³ are all the same.

In certain embodiments, at least one of R¹ and R² is selected fromC₁₋₆hydroxyalkyl and C₁₋₆alkoxyalkyl. In certain such embodiments, atleast one of R¹ and R² is alkoxyalkyl. In certain such embodiments, atleast one of R¹ and R² is selected from methoxymethyl and methoxyethyl.

In certain embodiments, R³ is selected from C₁₋₆alkyl and C₁₋₆aralkyl,preferably C₁₋₆alkyl. In certain such embodiments, R³ is selected frommethyl, ethyl, isopropyl, sec-butyl, and isobutyl. In certain suchembodiments R³ is isobutyl. In certain alternative embodiments, R³ isselected from phenylmethyl and phenylethyl, preferably phenylmethyl.

In certain embodiments, R⁴, R⁶, and R⁷ are independently selected fromhydrogen and methyl, preferably hydrogen.

In certain embodiments, R⁵ is a 5- or 6-membered heteroaryl. In certainsuch embodiments, R⁵ is selected from isoxazole, isothiazole, furan,thiophene, oxazole, thiazole, pyrazole, or imidazole, preferablyisoxazole, furan, or thiazole.

In certain embodiments, R⁵ is a bicyclic heteroaryl. In certain suchembodiments bicyclic heteroaryl is selected from benzisoxazole,benzoxazole, benzothiazole, benzisothiazole.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anisoxazol-3-yl or isoxazol-5-yl. In certain preferred such embodiments,when the isoxazol-3-yl is substituted, it is substituted at least at the5-position. In certain preferred embodiments, when the isoxazol-5-yl issubstituted, it is substituted at least at the 3-position.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anunsubstituted isoxazol-3-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substitutedisoxazol-3-yl. In certain such embodiments, R⁵ is isoxazol-3-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂-aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆-carbocycloalkyl. In certain preferredsuch embodiments R⁵ is isoxazole-3-yl substituted with a substituentselected from methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a 4- to 6-membered nitrogen-containingC₁₋₆heterocycloalkyl. In certain such embodiments, R⁵ is isoxazol-3-ylsubstituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. Incertain alternative such embodiments, L is C═O, Z is absent, and R⁵ isisoxazol-3-yl substituted with

wherein W is O, NR, or CH₂, and R is H or C₁₋₆alkyl. In certain suchembodiments, W is O.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with 5-membered nitrogen-containing C₁₋₆heteroaralkyl, suchas pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably1,2,4-triazol-5-ylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with C₁₋₆alkoxy or C₁₋₆alkoxyalkyl, preferably methoxy,ethoxy, methoxymethyl, or methoxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with C₁₋₆hydroxyalkyl, preferably hydroxymethyl orhydroxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂-aminocarboxylate, orC₁₋₆alkylcarboxylate. In certain such embodiments, R⁵ is substitutedwith methyl carboxylate or ethyl carboxylate, preferably methylcarboxylate.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anunsubstituted isoxazol-5-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substitutedisoxazol-5-yl. In certain such embodiments, R⁵ is isoxazol-5-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂-aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆-carbocycloalkyl In certain preferred suchembodiments R⁵ is isoxazole-3-yl substituted with a substituent selectedfrom methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a 4- to 6-membered nitrogen-containingC₁₋₆heterocycloalkyl. In certain such embodiments, R⁵ is isoxazol-5-ylsubstituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. Incertain alternative such embodiments, L is C═O, Z is absent, and R⁵ isisoxazol-3-yl substituted with

wherein W is O, NR, or CH₂, and R is H or C₁₋₆alkyl. In certain suchembodiments, W is O.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with 5-membered nitrogen-containing C₁₋₆heteroaralkyl, suchas pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably1,2,4-triazol-5-ylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with C₁₋₆alkoxy or C₁₋₆alkoxyalkyl, preferably methoxy,ethoxy, methoxymethyl, or methoxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with C₁₋₆hydroxyalkyl, preferably hydroxymethyl orhydroxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂-aminocarboxylate, orC₁₋₆alkylcarboxylate. In certain such embodiments, R⁵ is substitutedwith methyl carboxylate or ethyl carboxylate, preferably methylcarboxylate.

In certain embodiments, a compound of formula I is selected from

One aspect of the invention relates to a medical device includingcomposition disclosed herein that include an inhibitor having astructure of formula I. In one embodiment, the composition isincorporated within a medical device. In certain embodiments, themedical device is a gel comprising a polymer matrix or ceramic matrixand an inhibitor. Said polymer can be either naturally occurring orsynthetic. In another embodiment, said gel serves as a drug depot, anadhesive, a suture, a barrier or a sealant.

Another aspect of the invention relates to a medical device comprising asubstrate having a surface onto which an inhibitor having a structure offormula I is disposed. In one embodiment, the inhibitor is directlydisposed on a medical device. In another embodiment, a coating is sodisposed, the coating comprising a polymer matrix or ceramic matrix withan inhibitor having a structure of formula I dispersed or dissolvedtherein.

In one embodiment, the medical device is a coronary, vascular,peripheral, or biliary stent. More particularly, the stent of thepresent invention is an expandable stent. When coated with a matrixcontaining an inhibitor having a structure formula I, the matrix isflexible to accommodate compressed and expanded states of such anexpandable stent. In another embodiment of this invention, the stent hasat least a portion which is insertable or implantable into the body of apatient, wherein the portion has a surface which is adapted for exposureto body tissue and wherein at least a part of the surface is coated withan inhibitor having a structure of formula I, or a coating comprising amatrix having an inhibitor having a structure of formula I is dispersedor dissolved therein. An example of a suitable stent is disclosed inU.S. Pat. No. 4,733,665, which is incorporated herein by reference inits entirety.

In another embodiment, the medical device of the present invention is asurgical implement such as a vascular implant, an intraluminal device,surgical sealant or a vascular support. More particularly, the medicaldevice of the present invention is a catheter, an implantable vascularaccess port, a central venous catheter, an arterial catheter, a vasculargraft, an intraaortic balloon pump, a suture, a ventricular assist pump,a drug-eluting barrier, an adhesive, a vascular wrap, anextra/perivascular support, a blood filter, or a filter adapted fordeployment in a blood vessel, coated with an inhibitor having astructure of formula I either directly or by a matrix containing aninhibitor having a structure of formula I.

In certain embodiments, the intraluminal medical device is coated withan inhibitor having a structure of formula I or a coating comprisingbiologically tolerated matrix and an inhibitor having a structure offormula I dispersed in the polymer, said device having an interiorsurface and an exterior surface, having the coating applied to at leasta part of the interior surface, the exterior surface, or both.

In certain embodiments, the medical device may be useful to preventrestenosis after angioplasty. The medical device may also be useful forthe treatment of various diseases and conditions by providing localizedadministration of an inhibitor having a structure of formula I. Suchdiseases and conditions include restenosis, inflammation, rheumatoidarthritis, tissue injury due to inflammation, hyperproliferativediseases, severe or arthritic psoriasis, muscle-wasting diseases,chronic infectious diseases, abnormal immune response, conditionsinvolving vulnerable plaques, injuries related to ischemic conditions,and viral infection and proliferation. Examples of diseases andconditions that are subject to a treatment including the drug coatedmedical devices of the present invention include atherosclerosis, acutecoronary syndrome, Alzheimer's disease, cancer, fever, muscle disuse(atrophy), denervation, vascular occlusions, stroke, HIV infection,nerve injury, renal failure associated with acidosis, and hepaticfailure. See, e.g., Goldberg, U.S. Pat. No. 5,340,736.

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

As used herein, “enzyme” can be any partially or wholly proteinaceousmolecule which carries out a chemical reaction in a catalytic manner.Such enzymes can be native enzymes, fusion enzymes, proenzymes,apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinatedenzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linkedenzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurringor artificially-generated mutant enzymes, enzymes with side chain orbackbone modifications, enzymes having leader sequences, and enzymescomplexed with non-proteinaceous material, such as proteoglycans,proteoliposomes. Enzymes can be made by any means, including naturalexpression, promoted expression, cloning, various solution-based andsolid-based peptide syntheses, and similar methods known to those ofskill in the art.

The term “C₁₋₆heteroaralkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heteroaryl group.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example,tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine,lactones, lactams, and the like.

The term “C₁₋₆heterocycloalkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heterocyclyl group.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

As used herein, the term “inhibitor” is meant to describe a compoundthat blocks or reduces an activity of an enzyme (for example, inhibitionof proteolytic cleavage of standard fluorogenic peptide substrates suchas suc-LLVY-AMC, Box-LLR-AMC and Z-LLE-AMC, inhibition of variouscatalytic activities of the 20S proteasome). An inhibitor can act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitorcan bind reversibly or irreversibly, and therefore the term includescompounds that are suicide substrates of an enzyme. An inhibitor canmodify one or more sites on or near the active site of the enzyme, or itcan cause a conformational change elsewhere on the enzyme.

As used herein, the term “orally bioavailable” is meant to describe acompound administered to a mouse at 40 mg/kg or less, 20 mg/kg or less,or even 10 mg/kg or less, wherein one hour after oral administrationsuch a compound shows at least about 50%, at least about 75% or even atleast about 90% inhibition of proteasome CT-L activity in the blood.

As used herein, the term “peptide” includes not only standard amidelinkage with standard α-substituents, but commonly utilizedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, as detailed below.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” encompasses compounds that, under physiologicalconditions, are converted into therapeutically active agents. A commonmethod for making a prodrug is to include selected moieties that arehydrolyzed under physiological conditions to reveal the desiredmolecule. In other embodiments, the prodrug is converted by an enzymaticactivity of the host animal.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S-alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

Selectivity for 20S Proteasome

The enzyme inhibitors disclosed herein are useful in part because theyinhibit the action of the 20S proteasome. Additionally, unlike other 20Sproteasome inhibitors, the compounds disclosed herein are highlyselective toward the 20S proteasome, with respect to other proteaseenzymes. That is, the instant compounds show selectivities for the 20Sproteasome over other proteases such as cathepsins, calpains, papain,chymotrypsin, trypsin, tripeptidyl pepsidase II. The selectivities ofthe enzyme inhibitors for 20S proteasome are such that at concentrationsbelow about 50 μM, the enzyme inhibitors show inhibition of thecatalytic activity of the 20S proteasome, while not showing inhibitionof the catalytic activity of other proteases such as cathepsins,calpains, papain, chymotrypsin, trypsin, tripeptidyl pepsidase II. Inpreferred embodiments, the enzyme inhibitors show inhibition of thecatalytic activity of the 20S proteasome at concentrations below about10 μM, while not showing inhibition of the catalytic activity of otherproteases at these concentrations. In even more preferred embodiments,the enzyme inhibitors show inhibition of the catalytic activity of the20S proteasome at concentrations below about 1 μM, while not showinginhibition of the catalytic activity of other proteases at theseconcentrations. Enzyme kinetic assays are disclosed in U.S. applicationSer. No. 09/569,748, Example 2 and Stein et al., Biochem. (1996), 35,3899-3908.

Selectivity for Chymotrypsin-Like Activity

Particular embodiments of the enzyme inhibiting compounds describedherein are further useful because they can efficiently and selectivelyinhibit the chymotrypsin-like activity of the 20S proteasome, ascompared to the trypsin-like, and PGPH activities. The chymotrypsin-likeactivity of 20S proteasome is characterized by cleavage of peptides inthe immediate vicinity of large hydrophobic residues. In particular, thechymotrypsin-like activity of Ntn hydrolases can be determined bycleavage of a standard substrate. Examples of such substrates are knownin the art. For example, a leucylvalinyltyrosine derivative can be used.Enzyme kinetic assays are disclosed in U.S. application Ser. No.09/569,748, Example 2 and Stein et al., Biochem. (1996), 35, 3899-3908.

Uses of Enzyme Inhibitors

The biological consequences of proteasome inhibition are numerous.Proteasome inhibition has been suggested as a prevention and/ortreatment of a multitude of diseases including, but not limited to,proliferative diseases, neurotoxic/degenerative diseases, Alzheimer's,ischemic conditions, inflammation, immune-related diseases, HIV,cancers, organ graft rejection, septic shock, inhibition of antigenpresentation, decreasing viral gene expression, parasitic infections,conditions associated with acidosis, macular degeneration, pulmonaryconditions, muscle wasting diseases, fibrotic diseases, bone and hairgrowth diseases. Therefore, proteasome inhibitor compositions, such asthe orally bioavailable peptide epoxy ketone class of molecules asdescribed herein, provide a means of treating patients with theseconditions.

Proteasome inhibitor compositions may be used to treat conditionsmediated directly by the proteolytic function of the proteasome such asmuscle wasting, or mediated indirectly via proteins which are processedby the proteasome such as NF-κB. The proteasome participates in therapid elimination and post-translational processing of proteins (e.g.,enzymes) involved in cellular regulation (e.g., cell cycle, genetranscription, and metabolic pathways), intercellular communication, andthe immune response (e.g., antigen presentation). Specific examplesdiscussed below include β-amyloid protein and regulatory proteins suchas cyclins and transcription factor NF-κB.

At the cellular level, the accumulation of polyubiquitinated proteins,cell morphological changes, and apoptosis have been reported upontreatment of cells with various proteasome inhibitors. The proteasomedegrades many proteins in maturing reticulocytes and growingfibroblasts. In cells deprived of insulin or serum, the rate ofproteolysis nearly doubles. Inhibiting the proteasome reducesproteolysis, thereby reducing both muscle protein loss and thenitrogenous load on kidneys or liver. One aspect of the inventionrelates to the treatment of cachexia and muscle-wasting diseases.Compounds of the invention may be useful for treating conditions such ascancer, chronic infectious diseases, fever, muscle disuse (atrophy) anddenervation, nerve injury, fasting, renal failure associated withacidosis, and hepatic failure. See, e.g., Goldberg, U.S. Pat. No.5,340,736. Certain embodiments of the invention therefore encompasscompositions for: reducing the rate of muscle protein degradation in acell; reducing the rate of intracellular protein degradation; reducingthe rate of degradation of p53 protein in a cell; and inhibiting thegrowth of p53-related cancers. Each of these methods includes contactinga cell (in vivo or in vitro, e.g., a muscle in a subject) with aneffective amount of a pharmaceutical composition comprising a proteasomeinhibitor disclosed herein.

Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theimmune system screens for autologous cells that are virally infected orhave undergone oncogenic transformation. In certain embodiments, theinvention relates to a method for inhibiting antigen presentation in acell, comprising exposing the cell to a compound described herein.Proteasome inhibitors of the invention may be used to treatimmune-related conditions such as allergy, asthma, organ/tissuerejection (graft-versus-host disease), and auto-immune diseases,including, but not limited to, lupus, rheumatoid arthritis, psoriasis,multiple sclerosis, and inflammatory bowel diseases (such as ulcerativecolitis and Crohn's disease). Thus, in certain embodiments, theinvention relates to a method for suppressing the immune system of asubject comprising administering to the subject an effective amount of aproteasome inhibitor compound described herein.

In certain embodiments, the invention relates to a method for alteringthe repertoire of antigenic peptides produced by the proteasome or otherNtn with multicatalytic activity. For example, if the PGPH activity of20S proteasome is selectively inhibited, a different set of antigenicpeptides will be produced by the proteasome and presented in MHCmolecules on the surfaces of cells than would be produced and presentedeither without any enzyme inhibition, or with, for example, selectiveinhibition of chymotrypsin-like activity of the proteasome.

Another aspect of the invention relates to the use of proteasomeinhibitor compositions disclosed herein for the treatment ofneurodegenerative diseases and conditions, including, but not limitedto, stroke, ischemic damage to the nervous system, neural trauma (e.g.,percussive brain damage, spinal cord injury, and traumatic damage to thenervous system), multiple sclerosis and other immune-mediatedneuropathies (e.g., Guillain-Barre syndrome and its variants, acutemotor axonal neuropathy, acute inflammatory demyelinatingpolyneuropathy, and Fisher Syndrome), HIV/AIDS dementia complex,axonomy, diabetic neuropathy, Parkinson's disease, Huntington's disease,multiple sclerosis, bacterial, parasitic, fungal, and viral meningitis,encephalitis, vascular dementia, multi-infarct dementia, Lewy bodydementia, frontal lobe dementia such as Pick's disease, subcorticaldementias (such as Huntington or progressive supranuclear palsy), focalcortical atrophy syndromes (such as primary aphasia), metabolic-toxicdementias (such as chronic hypothyroidism or B12 deficiency), anddementias caused by infections (such as syphilis or chronic meningitis).

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which isidentical to the β-subunit of human macropain (Kojima, S. et al., Fed.Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing enzyme cleavesat the Gln¹⁵-Lys¹⁶ bond; in the presence of calcium ion, the enzyme alsocleaves at the Met-¹-Asp¹ bond, and the Asp¹-Ala² bonds to release theextracellular domain of β-AP.

One aspect of the invention, therefore, relates to a method of treatingAlzheimer's disease, comprising administering to a subject an effectiveamount of a proteasome inhibitor composition disclosed herein. Suchtreatment includes reducing the rate of β-AP processing, reducing therate of β-AP plaque formation, reducing the rate of β-AP generation, andreducing the clinical signs of Alzheimer's disease.

Fibrosis is the excessive and persistent formation of scar tissueresulting from the hyperproliferative growth of fibroblasts and isassociated with activation of the TGF-β signaling pathway. Fibrosisinvolves extensive deposition of extracellular matrix and can occurwithin virtually any tissue or across several different tissues.Normally, the level of intracellular signaling protein (Smad) thatactivate transcription of target genes upon TGF-β stimulation isregulated by proteasome activity (Xu et al., 2000). However, accelerateddegradation of the TGF-β signaling components has been observed incancers and other hyperproliferative conditions. Thus, in certainembodiments the invention relates to a method for treatinghyperproliferative conditions such as diabetic retinopathy, maculardegeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy,cirrhosis, biliary atresia, congestive heart failure, scleroderma,radiation-induced fibrosis, and lung fibrosis (idiopathic pulmonaryfibrosis, collagen vascular disease, sarcoidosis, interstitial lungdiseases and extrinsic lung disorders). The treatment of burn victims isoften hampered by fibrosis, thus, in certain embodiments, the inventionrelates to the topical or systemic administration of the inhibitors totreat burns. Wound closure following surgery is often associated withdisfiguring scars, which may be prevented by inhibition of fibrosis.Thus, in certain embodiments, the invention relates to a method for theprevention or reduction of scarring.

Certain proteasome inhibitors block both degradation and processing ofubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors alsoblock IκB-A degradation and NF-κB activation (Palombella, et al. Cell(1994) 78:773-785; and Traenckner, et al., EMBO J. (1994) 13:5433-5441).One aspect of the invention relates to a method for inhibiting IκB-αdegradation, comprising contacting the cell with a compound describedherein. In certain embodiments, the invention relates to a method forreducing the cellular content of NF-κB in a cell, muscle, organ, orsubject, comprising contacting the cell, muscle, organ, or subject witha proteasome inhibitor compound described herein.

NF-κB is a member of the Rel protein family. The Rel family oftranscriptional activator proteins can be divided into two groups. Thefirst group requires proteolytic processing, and includes p50 (NF-κB1,105 kDa) and p52 (NF-κ2, 100 kDa). The second group does not requireproteolytic processing, and includes p65 (RelA, Rel (c-Rel), and RelB).Both homo- and heterodimers can be formed by Rel family members; NF-κB,for example, is a p50-p65 heterodimer. After phosphorylation andubiquitination of IκB and p105, the two proteins are degraded andprocessed, respectively, to produce active NF-κB which translocates fromthe cytoplasm to the nucleus. Ubiquitinated p105 is also processed bypurified proteasomes (Palombella et al., Cell (1994) 78:773-785). ActiveNF-κB forms a stereospecific enhancer complex with other transcriptionalactivators and, e.g., HMG I(Y), inducing selective expression of aparticular gene.

NF-κB regulates genes involved in the immune and inflammatory response,and mitotic events. For example, NF-κB is required for the expression ofthe immunoglobulin light chain κ gene, the IL-2 receptor α-chain gene,the class I major histocompatibility complex gene, and a number ofcytokine genes encoding, for example, IL-2, IL-6, granulocytecolony-stimulating factor, and IFN-β (Palombella et al., Cell (1994)78:773-785). In certain embodiments, the invention relates to methods ofaffecting the level of expression of IL-2, MHC-I, IL-6, TNFα, IFN-β orany of the other previously-mentioned proteins, each method comprisingadministering to a subject an effective amount of a proteasome inhibitorcomposition disclosed herein. Complexes including p50 are rapidmediators of acute inflammatory and immune responses (Thanos, D. andManiatis, T., Cell (1995) 80:529-532).

Overproduction of lipopolysaccharide (LPS)-induced cytokines such asTNFα is considered to be central to the processes associated with septicshock. Furthermore, it is generally accepted that the first step in theactivation of cells by LPS is the binding of LPS to specific membranereceptors. The α- and β-subunits of the 20S proteasome complex have beenidentified as LPS-binding proteins, suggesting that the LPS-inducedsignal transduction may be an important therapeutic target in thetreatment or prevention of sepsis (Qureshi, N. et al., J. Immun. (2003)171: 1515-1525). Therefore, in certain embodiments, the proteasomeinhibitor compositions may be used for the inhibition of TNFα to preventand/or treat septic shock.

NF-κB also participates in the expression of the cell adhesion genesthat encode E-selectin, P-selectin, ICAM, and VCAM-1 (Collins, T., Lab.Invest. (1993) 68:499-508). In certain embodiments the invention relatesto a method for inhibiting cell adhesion (e.g., cell adhesion mediatedby E-selectin, P-selectin, ICAM, or VCAM-1), comprising contacting acell with (or administering to a subject) an effective amount of apharmaceutical composition comprising a proteasome inhibitor disclosedherein.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of IκB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV (Cohen, J.,Science, (1995) 267:960). In certain embodiments, the invention relatesto a method for inhibiting or reducing HIV infection in a subject, or amethod for decreasing the level of viral gene expression, each methodcomprising administering to the subject an effective amount of aproteasome inhibitor composition disclosed herein.

Viral infections contribute to the pathology of many diseases. Heartconditions such as ongoing myocarditis and dilated cardiomyopathy havebeen linked to the coxsackievirus B3. In a comparative whole-genomemicroarray analyses of infected mouse hearts, specific proteasomesubunits were uniformly up-regulated in hearts of mice which developedchronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Someviruses utilize the ubiquitin-proteasome system in the viral entry stepwhere the virus is released from the endosome into the cytosol. Themouse hepatitis virus (MHV) belongs to the Coronaviridae family, whichalso includes the severe acute respiratory syndrome (SARS) coronvirus.Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment ofcells infected with MHV with a proteasome inhibitor resulted in adecrease in viral replication, correlating with reduced viral titer ascompared to that of untreated cells. The human hepatitis B virus (HBV),a member of the Hepadnaviridae virus family, likewise requires virallyencoded envelop proteins to propagate. Inhibiting the proteasomedegradation pathway causes a significant reduction in the amount ofsecreted envelope proteins (Simsek et al, J Virol 79:12914-12920, 2005).In addition to HBV, other hepatitis viruses (A, C, D and E) may alsoutilize the ubiquitin-proteasome degradation pathway for secretion,morphogenesis and pathogenesis. Accordingly, in certain embodiments, theinvention relates to a method for treating viral infection, such as SARSor hepatitis A, B, C, D and E, comprising contacting a cell with (oradministering to a subject) an effective amount of a compound disclosedherein.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-Bα, thereby resulting inthe activation of NF-κB (Koong et al., 1994). It has been demonstratedthat the severity of injury resulting in hypoxia can be reduced with theadministration of a proteasome inhibitor (Gao et al., 2000; Bao et al.,2001; Pye et al., 2003). Therefore, certain embodiments of the inventionrelate to a method of treating an ischemic condition or reperfusioninjury comprising administering to a subject in need of such treatmentan effective amount of a proteasome inhibitor compound disclosed herein.Examples of such conditions or injuries include, but are not limited to,acute coronary syndrome (vulnerable plaques), arterial occlusive disease(cardiac, cerebral, peripheral arterial and vascular occlusions),atherosclerosis (coronary sclerosis, coronary artery disease),infarctions, heart failure, pancreatitis, myocardial hypertrophy,stenosis, and restenosis.

Other eukaryotic transcription factors that require proteolyticprocessing include the general transcription factor TFIIA, herpessimplex virus VP16 accessory protein (host cell factor), virus-inducibleIFN regulatory factor 2 protein, and the membrane-bound sterolregulatory element-binding protein 1.

In certain embodiments, the invention relates to methods for affectingcyclin-dependent eukaryotic cell cycles, comprising exposing a cell (invitro or in vivo) to a proteasome inhibitor composition disclosedherein. Cyclins are proteins involved in cell cycle control. Theproteasome participates in the degradation of cyclins. Examples ofcyclins include mitotic cyclins, G1 cyclins, and cyclin B. Degradationof cyclins enables a cell to exit one cell cycle stage (e.g., mitosis)and enter another (e.g., division). It is believed all cyclins areassociated with p34^(cdc2) protein kinase or related kinases. Theproteolysis targeting signal is localized to amino acids42-RAALGNISEN-50 (destruction box). There is evidence that cyclin isconverted to a form vulnerable to a ubiquitin ligase or that acyclin-specific ligase is activated during mitosis (Ciechanover, A.,Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclindegradation, and therefore inhibits cell proliferation, for example, incyclin-related cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA(1990) 87:7071-7075). One aspect of the invention relates to a methodfor treating a proliferative disease in a subject (e.g., cancer,psoriasis, or restenosis), comprising administering to the subject aneffective amount of a proteasome inhibitor composition disclosed herein.The invention also relates to a method for treating cyclin-relatedinflammation in a subject, comprising administering to a subject atherapeutically effective amount of a proteasome inhibitor compositiondescribed herein.

Additional embodiments of the invention relate to methods for affectingthe proteasome-dependent regulation of oncoproteins and methods oftreating or inhibiting cancer growth, each method comprising exposing acell (in vivo, e.g., in a subject, or in vitro) to a proteasomeinhibitor composition disclosed herein. HPV-16 and HPV-18-derived E6proteins stimulate ATP- and ubiquitin-dependent conjugation anddegradation of p53 in crude reticulocyte lysates. The recessive oncogenep53 has been shown to accumulate at the nonpermissive temperature in acell line with a mutated thermolabile E1. Elevated levels of p53 maylead to apoptosis. Examples of proto-oncoproteins degraded by theubiquitin system include c-Mos, c-Fos, and c-Jun. In certainembodiments, the invention relates to a method for treating p53-relatedapoptosis, comprising administering to a subject an effective amount ofa proteasome inhibitor composition disclosed herein.

In certain embodiments, the disclosed compositions may be useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, theadministrative protocols for the proteasome inhibitor compositions areuseful for the treatment of parasitic infections in humans caused by aprotozoan parasite selected from Plasmodium sps. (including P.falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria),Trypanosoma sps. (including T. cruzi, which causes Chagas' disease, andT. brucei which causes African sleeping sickness), Leishmania sps.(including L. amazonesis, L. donovani, L. infantum, L. mexicana, etc.),Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS andother immunosuppressed patients), Toxoplasma gondii, Entamoebahistolytica, Entamoeba invadens, and Giardia lamblia. In certainembodiments, the disclosed proteasome inhibitor compositions are usefulfor the treatment of parasitic infections in animals and livestockcaused by a protozoan parasite selected from Plasmodium hermani,Cryptosporidium sps., Echinococcus granulosus, Eimeria tenella,Sarcocystis neurona, and Neurospora crassa. Other compounds useful asproteasome inhibitors in the treatment of parasitic diseases aredescribed in WO 98/10779, which is incorporated herein in its entirety.

In certain embodiments, the proteasome inhibitor compositions inhibitproteasome activity in a parasite without recovery in red blood cellsand white blood cells. In certain such embodiments, the long half-lifeof blood cells may provide prolonged protection with regard to therapyagainst recurring exposures to parasites. In certain embodiments, theproteasome inhibitor compositions may provide prolonged protection withregard to chemoprophylaxis against future infection.

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed proteasome inhibitor compositions may be useful in thetreatment and/or prevention of diseases associated with bone loss, suchas osteroporosis.

Proteasome inhibition has already been validated as a therapeuticstrategy for the treatment of cancer, particularly multiple myeloma.However, based on both in vitro and in vivo models, one would predictthat it could serve as a strategy against other cancers, particularlyheme-related malignancies and solid tumors. Therefore, certainembodiments of the invention relate to a method of treating cancerscomprising administering to a subject in need of such treatment aneffective amount of a proteasome inhibitor compound disclosed herein.

Administration

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteasomeinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential, or separate dosing of the individualcomponents of the treatment.

In certain embodiments, a compound of the invention is conjointlyadministered with one or more other proteasome inhibitor(s).

In certain embodiments, a compound of the invention is conjointlyadministered with a chemotherapeutic. Suitable chemotherapeutics mayinclude, natural products such as vinca alkaloids (i.e. vinblastine,vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e.etoposide, teniposide), antibiotics (dactinomycin (actinomycin D)daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone,bleomycins, plicamycin (mithramycin) and mitomycin, enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates(busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin),trazenes-dacarbazinine (DTIC); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate), pyrimidineanalogs (fluorouracil, floxuridine, and cytarabine), purine analogs andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane,and letrozole); and platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;histone deacetylase (HDAC) inhibitors; hormones (i.e. estrogen) andhormone agonists such as leutinizing hormone releasing hormone (LHRH)agonists (goserelin, leuprolide and triptorelin). Other chemotherapeuticagents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen,raloxifene, gemcitabine, navelbine, or any analog or derivative variantof the foregoing.

In certain embodiments, a compound of the invention is conjointlyadministered with a cytokine. Cytokines include, but are not limited to,Interferon-γ, -α, and -β, Interleukins 1-8, 10 and 12, GranulocyteMonocyte Colony-Stimulating factor (GM-CSF), TNF-α and -β, and TGF-β.

In certain embodiments, a compound of the invention is conjointlyadministered with a steroid. Suitable steroids may include, but are notlimited to, 21-acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, meprednisone, methylprednisolone,mometasone furoate, paramethasone, prednicarbate, prednisolone,prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate,prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide,triamcinolone hexacetonide, and salts and/or derivatives thereof.

In certain embodiments, a compound of the invention is conjointlyadministered with an immunotherapeutic agent. Suitable immunotherapeuticagents may include, but are not limited to, MDR modulators (verapamil,valspordar, biricodar, tariquidar, laniquidar), rapamycin, mycophenylatemofetil, cyclophosomide, cyclosporine, thalidomide, and monoclonalantibodies. The monoclonal antibodies can be either naked or conjugatedsuch as rituximab, tositumomab, alemtuzumab, daclizumab, epratuzumab,ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab,erlotinib and trastuzumab.

EXEMPLIFICATION Example 1

Compound (003):

To a 0° C. solution of N-Boc serine(methyl ether) (001) (2.5 g, 11.4mmol), L-alanine benzyl ester hydrochloride (002) (3.3 g, 11.4 mmol),HOBT (2.5 g, 18.2 mmol) and HBTU (6.9 g, 18.24 mmol) in tetrahydrofuran(400 mL) was added a solution of N,N-diisopropylethylamine (8.0 mL, 45.6mmol) in tetrahydrofuran (50 mL) over 10 minutes. The mixture wasstirred at room temperature for another 5 hours. Most of the solventswere removed under reduced pressure and the resulting material dilutedwith ethyl acetate (300 mL). The solution was then washed with saturatedaqueous sodium bicarbonate (2×50 mL) and brine (100 mL). The organiclayers were dried over sodium sulfate and filtered through Celite-545.The solvents were removed under reduced pressure and the residue waspurified by flash chromatography (hexane and ethyl acetate), and thedesired compound (003) (4.4 g) was isolated and characterized by LC/MS(LCRS (MH) m/z: 457.23).

Compound (004):

To a 0° C. solution of (003) (5.14 g, 11.25 mmol) in tetrahydrofuran(100 mL) was added 10% Pd/C (500 mg). The resulting mixture was allowedto stir under 1 atmosphere of hydrogen for 4 hours. The mixture wasfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The organic filtrate was concentrated under reducedpressure and placed under high vacuum for 2 hours to provide (004) asconfirmed by LC/MS (LCRS (MH) m/z: 367.18) which was used withoutfurther purification.

Compound (006):

To a solution of (005) (for a synthesis of (005) see U.S. patentapplication Ser. No. 11/131,688) (3.9 g, 13 mmol) in trifluoroaceticacid (50 mL) was added 10% Pd/C (600 mg). The resulting mixture wasallowed to stir under 1 atmosphere of hydrogen for 6 hours. The mixturewas filtered through Celite-545 and the filter cake washed withdichloromethane (200 mL). The filtrate was concentrated under reducedpressure and placed under high vacuum overnight to provide (006) asconfirmed by LC/MS (LCRS (MH) m/z: 172.13) and was used in thesubsequent transformation without further purification.

Compound (007):

To a 0° C. solution of (004) and (006), HOBT (2.5 g, 18 mmol) and HBTU(6.9 g, 18 mmol) in tetrahydrofuran (400 mL) was added a solution ofN,N-diethylisopropylamine (8 mL, 46 mmol) in tetrahydrofuran (50 mL)over 10 minutes. The mixture was stirred at room temperature for another5 hours. Most of the solvents were removed under reduced pressure andthe remaining material was diluted with ethyl acetate (400 mL). Theresulting solution was washed with saturated aqueous sodium bicarbonate(2×100 mL) and brine (100 mL). The organic layers were dried over sodiumsulfate and filtered through Celite-545. The solvents were removed underreduced pressure and the residue was purified by flash chromatography(hexane and ethyl acetate), to provide (007) (3.5 g) as characterized byLC/MS (LCRS (MH) m/z: 520.29).

Compound (008):

To a 0° C. solution of (007) (320 mg, 0.616 mmol) in dichloromethane (10mL) was added trifluoroacetic acid (10 mL) and the resulting solutionwas stirred at the same temperature for another hour. The organic layerswere concentrated under reduced pressure and then placed under highvacuum for 2 hours to provide (008) as confirmed by LC/MS (LCRS (MH)m/z: 420.24) which was used without further purification.

Compound (010):

To a 0° C. solution of (008), 5-methyl-isoxazole-3-carboxylic acid (009)(94 mg, 0.74 mmol), HOBT (135 mg, 1.0 mmol) and HBTU (350 mg, 1.0 mmol)in tetrahydrofuran (100 mL) was added a solution ofN,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrahydrofuran (2 mL)over 5 minutes. The mixture was stirred at room temperature for another5 hours and then diluted with ethyl acetate (200 mL). It was then washedwith saturated aqueous sodium bicarbonate (2×10 mL) and brine (10 mL)and the organic layers were dried over sodium sulfate and filteredthrough Celite-545. The solvents were removed under reduced pressure andthe residue was purified by HPLC (aqueous ammonium acetate andacetonitrile) to provide (010) (195 mg) as characterized by LC/MS (LCRS(MH) m/z: 529.26); >90% proteasome CT-L inhibition at 40 mg/kg PO.

Example 2

Compound (013):

To a 0° C. solution of N-Boc L-leucine (011) (2.6 g, 11 mmol),L-phenylalanine benzyl ester hydrochloride (012) (2.9 g, 10 mmol), HOBT(1.7 g, 11 mmol) and HBTU (3.9 g, 11 mmol) in tetrahydrofuran (200 mL)was added a solution of N,N-diisopropylethylamine (4.9 mL, 30 mmol) intetrahydrofuran (10 mL) over 5 minutes. The mixture was stirred at roomtemperature for another 5 hours and became homogenous. It was thendiluted with ethyl acetate (300 mL) and washed with saturated aqueoussodium bicarbonate (2×50 mL) and brine (100 mL). The organic layers weredried over sodium sulfate and filtered through Celite-545. The solventswere removed under reduced pressure and the residue was purified byflash chromatography (hexane and ethyl acetate) to provide (013) (4.4 g)as characterized by LC/MS (LCRS (MH) m/z: 469.26).

Compound (014):

To a 0° C. solution of (013) (4.32 g, 9.24 mmol) in tetrahydrofuran (100mL) was added 10% Pd/C (500 mg). The resulting mixture was allowed tostir under 1 atmosphere of hydrogen for 4 hours. The mixture wasfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The organic filtrate was concentrated under reducedpressure and placed under high vacuum to provide (014) (3.5 g) asconfirmed by LC/MS (LCRS (MH) m/z: 378.22) which was used withoutfurther purification.

Compound (015):

To a 0° C. solution of (014) (3.5 g, 9.24 mmol) and (006) (2.4 g, 11mmol), HOBT (1.7 g, 11 mmol) and HBTU (3.9 g, 11 mmol) intetrahydrofuran (200 mL) was added a solution ofN,N-diisopropylethylamine (4.9 mL, 30 mmol) in tetrahydrofuran (10 mL)over 5 minutes. The mixture was stirred at room temperature for another5 hours and became homogenous. It was then diluted with ethyl acetate(400 mL), and washed with saturated aqueous sodium bicarbonate (2×100mL) and brine (100 mL). The organic layers were dried over sodiumsulfate and filtered through Celite-545. The solvents were removed underreduced pressure and the residue was purified by flash chromatography(hexane and ethyl acetate), and the desired compound (015) (5.0 g) wasisolated and characterized by LC/MS (LCRS (MH) m/z: 532.33).

Compound (016):

To a 0° C. solution of (015) (5.0 g, 9.40 mmol) in dichloromethane (50mL) was added trifluoroacetic acid (20 mL) over 5 minutes, and theresulting solution was stirred at the same temperature for another hour.The organic layers were concentrated under reduced pressure and placedunder high vacuum to provide (016) as confirmed by LC/MS (LCRS (MH) m/z:432.33) which was used without further purification.

Compound (018):

To a solution of methyl 5-methyl-3-isoxazolecarboxylate (017) (14.1 g,100 mmol) in carbon tetrachloride (500 mL) was added N-bromosuccunimide(23 g, 130 mmol) and benzoyl peroxide (2.5 g, 10 mmol) at roomtemperature. The resulting mixture was stirred at 80° C. under anatmosphere of argon overnight. The reaction was the cooled and dilutedwith 500 mL of dichloromethane and washed with saturated aqueous sodiumbicarbonate (3×100 mL). The aqueous phase was extracted with 200 mL ofdichloromethane, and the combined organic layers were washed with brineand dried over MgSO₄. The solvents were removed and the residue waspurified by flash chromatography (hexane and ethyl acetate) to provide(018) (7.9 g) which was characterized by LC/MS (LCRS (MH) m/z: 219.95).

Compound (019):

To a 0° C. solution of (018) (12 g, 55 mmol) in tetrahydrofuran (20 mL)was added aqueous lithium hydroxide (35 mL, 4N). The resulting mixturewas stirred at room temperature overnight. It was then acidified withhydrochloric acid (2N) to pH=1 and extracted with tetrahydrofuran (3×200mL). The combine organic layers were washed with brine (10 mL), driedover sodium sulfate, and filtered. The solvents were removed and theresidue was lyophilized to yield (019) (8.2 g), which was confirmed byLC/MS (LCRS (MH) m/z: 205.95) and used without further purification.

Compound (020):

A solution of (019) (6.0 g, 30 mmol), benzyl alcohol (3.5 mL), andp-toluenesulfonyl acid (1.1 g, 6 mmol) in toluene (100 mL) was stirredat 100° C. overnight. It was then allowed to cool, diluted with 300 mLof ethyl acetate and washed with saturated aqueous sodium bicarbonate.The aqueous phase was then extracted with 200 mL of ethyl acetate. Thecombine organic layers were washed with brine, dried over sodiumsulfate, and filtered. The solvents were removed and the residue waspurified by flash chromatography (hexane and ethyl acetate), to provide(020) (5.8 g) which was characterized by LC/MS (LCRS (MH) m/z: 295.98).

Compound (021):

A solution of (020) (2.0 g, 6.8 mmol) and morpholine (3.0 mL) intetrahydrofuran (50 mL) was stirred at room temperature for two hours.The solvents were then removed and the residue was purified by flashchromatography (hexane and ethyl acetate/methanol) to provide (021) (820mg) which was characterized by LC/MS (LCRS (MH) m/z: 303.13).

Compound (022):

To a solution of (021) (400 mg, 1.32 mmol) in tetrahydrofuran (40 mL)was added 10% Pd/C (100 mg) and the mixture was stirred at roomtemperature under one atmosphere of hydrogen for 2 hours. It was thenfiltered through Celite and concentrated to give (022), which wasconfirmed by LC/MS (LCRS (MH) m/z: 213.08) and was used without furtherpurification.

Compound (023):

To a 0° C. solution of (016) (130 mg, 0.3 mmol) and (022) (70 mg, 0.4mmol), HOBT (70 mg, 0.5 mmol) and HBTU (170 mg, 0.5 mmol) intetrahydrofuran (50 mL) was added a solution ofN,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrahydrofuran (5 mL).The mixture was stirred at room temperature for another 5 hours andbecame homogenous. It was then diluted with ethyl acetate (200 mL) andwashed with saturated aqueous sodium bicarbonate (2×10 mL) and brine (10mL). The organic layers were dried over sodium sulfate and filteredthrough Celite-545. The solvents were removed under reduced pressure andthe residue was purified by HPLC (aqueous ammonium acetate andacetonitrile) to provide compound (023) (125 mg) which was characterizedby LC/MS (LCRS (MH) m/z: 626.35); >80% proteasome CT-L inhibition at 40mg/kg PO.

Example 3

Compound (025):

To a 0° C. solution of Fmoc-Val-OH (024) (348 mg, 1.6 mmol) indichloromethane (4 mL) were added MSNT (474 mg, 1.6 mmol) andN-methyl-imidazole (0.13 mL, 1.6 mmol). HMPB resin (400 mg, 0.32 mmol)was added once the mixture became homogenous. The resulting reactionmixture was allowed to shake for two hours at room temperature. Theresin was filtered off, washed with N,N-dimethylformamide (3×10 mL) anddichloromethane (3×10 mL), and was allowed to air dry to yield (025).

Compound (026):

Resin (025) was placed in a solution of 20% piperidine inN,N-dimethylformamide (20 ml) and the resulting mixture was shaken atroom temperature for 1 hour. The resin was filtered off and washed withN,N-dimethylformamide (3×10 mL) and dichloromethane (3×10 mL).

To a 0° C. solution of Fmoc-Ser(OMe)-OH (546 mg, 1.6 mmol) inN,N-dimethylformamide (4 mL) were added HOBT (245 mg, 1.6 mmol), HBTU(606 mg, 1.6 mmol,) and N,N-diisopropylethylamine (0.6 mL, 3.2 mmol).The resin was added once the reaction mixture became homogenous. Theresulting mixture was allowed to shake overnight. The resin was thenfiltered off and washed with N,N-dimethylformamide (3×10 mL) anddichloromethane (3×10 mL), and allowed to air dry to yield (026).

Compound (027):

Resin (026) was placed in a solution of 20% piperidine inN,N-dimethylformamide (20 mL) and the resulting mixture was shaken atroom temperature for 1 hour. The resin was filtered off and washed withN,N-dimethylformamide (3×10 mL) and dichloromethane (3×10 mL).

To a 0° C. solution of 5-methyl-isoxazole-3-carboxylic acid (009) (162mg, 1.6 mmol) in N,N-dimethylformamide (4 mL) were added HOBT (245 mg,1.6 mmol), HBTU (606 mg, 1.6 mmol) and N,N-diisopropylethylamine (0.6mL, 3.2 mmol). Once the resulting mixture became homogenous, the resinwas added and the resulting reaction mixture was allowed to shakeovernight. The resin was then filtered off and washed withN,N-dimethylformamide (3×10 mL) and dichloromethane (3×10 mL), and theresin was allowed to air dry to yield (027).

Compound (028):

To resin (027) was added a solution of 50% trifluoroacetic acid indichloromethane (10 mL), and the resulting mixture was allowed to shakefor 30 minutes. The resin was then filtered off and washed withdichloromethane (3×10 mL). The volatiles were removed under reducedpressure to provide (028) which was characterized by LC/MS (LCRS (MH)m/z: 328.14) and used without further purification.

Compound (029):

To a 0° C. solution of (029) and (006) (117 mg, 0.4 mmol), HOBT (70 mg,0.5 mmol) and HBTU (170 mg, 0.5 mmol) in tetrahydrofuran (50 mL) wasadded a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) intetrahydrofuran (5 mL). The mixture was stirred at room temperature foranother 5 hours and became homogenous. It was then diluted with ethylacetate (200 mL) and washed with saturated aqueous sodium bicarbonate(2×10 mL) and brine (10 mL). The organic layers were dried over sodiumsulfate and filtered through Celite-545. The solvents were removed underreduced pressure and the residue was purified by HPLC (aqueous ammoniumacetate and acetonitrile) to provide (029) (125 mg) which wascharacterized by LC/MS (LCRS (MH) m/z: 481.26); >70% proteasome CT-Linhibition at 20 mg/kg PO.

Example 4

Compound (031):

To a 0° C. solution of Fmoc-L-4-thiazolylalanine (030) (1.0 g, 2.5 mmol)in dichloromethane (4 mL) were added N-methyl-imidazole (150 uL, 1.9mmol), MSNT (755 mg, 2.55 mmol) and HMPB resin (800 mg, 0.51 mmol) wasadded once the mixture became homogenous. The resulting reaction mixturewas allowed to shake for two hours at room temperature. The resin wasthen filtered off and washed with N,N-dimethylformamide (3×20 mL) anddichloromethane (3×20 mL), and allowed to air dry to yield (031).

Compound (032):

Resin (031) (360 mg, 0.23 mmol) was placed in a solution of 20%piperidine in N,N-dimethylformamide (20 mL) and the resulting mixturewas shaken at room temperature for 1 hour. The resin was then filteredoff and washed with N,N-dimethylformamide (3×10 mL) and dichloromethane(3×10 mL).

To a 0° C. solution of Fmoc-L-Leucine (204 mg, 0.58 mmol) inN,N-dimethylformamide (4 mL) were added HOBT (124 mg, 0.92 mmol), HBTU(349 mg 0.92 mmol) and N,N-diisopropylethylamine (402 uL, 2.3 mmol). Theresin was then added once the reaction mixture became homogenous. Theresulting mixture was allowed to shake at 5° C. for five 5 hours. Theresin was then filtered off and washed with N,N-dimethylformamide (3×20mL) and dichloromethane (3×20 mL), and the resulting resin was allowedto air dry to yield (032).

Compound (033):

Resin (032) (0.23 mmol) was placed in a solution of 20% piperidine inN,N-dimethylformamide (20 mL) and the resulting mixture was shaken atroom temperature for 1 hour. The resin was then filtered off and washedwith N,N-dimethylformamide (3×10 mL) and dichloromethane (3×10 mL).

To a 0° C. solution of (022) (123 mg 0.58 mmol) in N,N-dimethylformamide(4 mL) were added HOBT (124 mg, 0.92 mmol), HBTU (349 mg 0.92 mmol) andN,N-diisopropylethylamine (402 uL, 2.3 mmol). Once the resulting mixturebecame homogenous, the resin was added and the resulting reactionmixture was allowed to shake at room temperature overnight. The resinwas then filtered off, washed with N,N-dimethylformamide (3×10 mL) anddichloromethane (3×10 mL), and the resulting resin was allowed to airdry to yield (033).

Compound (034):

To resin (033) was added a solution of 50% of trifluoroacetic acid indichloromethane (10 mL), and the resulting mixture was allowed to shakefor 30 minutes. It was then filtered off and the resin washed withdichloromethane (3×10 mL). The volatiles were removed under reducedpressure to provide (34) as characterized by LC/MS (LCRS (MH) m/z:480.18) which was used without further purification.

Compound (035):

To a 0° C. solution of (034) and (006) (70 mg, 0.23 mmol), HOBT (50 mg,0.37 mmol) and HBTU (140 mg, 0.37 mmol) in tetrahydrofuran (50 mL) wasadded a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) intetrahydrofuran (5 mL). The mixture was stirred at room temperature for5 hours and then diluted with ethyl acetate (200 mL). It was then washedwith saturated aqueous sodium bicarbonate (2×10 mL) and brine (10 mL).The organic layers were dried over sodium sulfate and filtered throughCelite-545 and the solvents were removed under reduced pressure. Theresulting residue was purified by HPLC (aqueous ammonium acetate andacetonitrile) to provide (035) (15 mg) which was characterized by LC/MS(LCRS (MH) m/z: 633.3); >90% proteasome CT-L inhibition at 40 mg/kg PO.

Example 5

Compound (036):

Resin (031) (800 mg, 0.23 mmol) was placed in a solution of 20%piperidine in N,N-dimethylformamide (20 mL) and the resulting mixturewas shaken at room temperature for 1 hour. The resin was filtered offand washed with N,N-dimethylformamide (3×10 mL) and dichloromethane(3×10 mL).

To a 0° C. solution of Fmoc-L-Ser(OMe)-OH (435 mg, 1.3 mmol) inN,N-dimethylformamide (10 mL) were added HOBT (276 mg, 2.0 mmol), HBTU(710 mg, 2.0 mmol) and N,N-diisopropylethylamine (0.9 mL, 5.1 mmol). Theresin was then added once the reaction mixture became homogenous. Theresulting mixture was allowed to shake at 5° C. for 5 hours. The resinwas then filtered off and washed with N,N-dimethylformamide (3×20 mL)and dichloromethane (3×20 mL) and allowed to air dry to yield (036).

Compound (037):

Resin (036) was placed in a solution of 20% piperidine inN,N-dimethylformamide (20 mL) and the resulting mixture was shaken atroom temperature for 1 hour. The resin was filtered off and washed withN,N-dimethylformamide (3×20 mL) and dichloromethane (3×20 mL).

To a 0° C. solution of (009) (162 mg, 1.3 mmol) in N,N-dimethylformamide(4 mL) were added HOBT (276 mg, 2.0 mmol), HBTU (710 mg, 2.0 mmol) andN,N-diisopropylethylamine (0.9 mL, 5.1 mmol). Once the resulting mixturebecame homogenous, the resin was added and the resulting reactionmixture was allowed to shake at room temperature overnight. The resinwas then filtered off, washed with N,N-dimethylformamide (3×10 mL) anddichloromethane (3×10 mL), and allowed to air dry to yield (037).

Compound (038):

To (037) was added a solution of 50% of trifluoroacetic acid indichloromethane (10 mL), and the resulting mixture was allowed to shakefor 30 minutes. The resin was then filtered off and washed withdichloromethane (3×10 mL). The volatiles were removed under reducedpressure to provide (38) which was characterized by LC/MS (LCRS (MH)m/z: 383.09) and used without further purification.

Compound (039):

To a 0° C. solution of (038) and (006) (156 mg, 0.51 mmol), HOBT (111mg, 0.82 mmol) and HBTU (311 mg, 0.82 mmol) in tetrahydrofuran (50 mL)was added a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) intetrahydrofuran (5 mL). The mixture was stirred at room temperature for5 hours and became homogenous. It was then diluted with ethyl acetate(200 mL) and washed with saturated aqueous sodium bicarbonate (2×10 mL)and brine (10 mL). The organic layers were dried over sodium sulfate andfiltered through Celite-545. The solvents were removed under reducedpressure and the residue was purified by HPLC (aqueous ammonium acetateand acetonitrile) to provide (039) (22 mg) which was characterized byLC/MS (LCRS (MH) m/z: 536.21); >75% proteasome CT-L inhibition at 20mg/kg PO.

Example 6

Compound (041):

To a 0° C. solution of Fmoc-L-alanine (040) (1.0 g, 3.2 mmol) indichloromethane (30 mL) were added N-methyl-imidazole (190 μL, 12.4mmol), MSNT (950 mg, 3.2 mmol), and HMPB resin (1.0 g, 0.64 mmol) whichwas then added once mixture became homogenous. The resulting reactionmixture was allowed to shake for two hours at room temperature. Theresin was then filtered off and washed with N,N-dimethylformamide (3×20mL) and dichloromethane (3×20 mL) to yield (041).

Compound (042):

Resin (041) was placed in a solution of 20% piperidine inN,N-dimethylformamide (20 mL) and the resulting mixture was shaken atroom temperature for 1 hour. The resin was filtered off and washed withN,N-dimethylformamide (3×20 mL) and dichloromethane (3×20 mL).

To a 0° C. solution of Fmoc-Ser(OMe)-OH (546 mg, 1.6 mmol) inN,N-dimethylformamide (10 mL) were added HOBT (346 mg, 2.6 mmol), HBTU(970 mg 2.6 mmol) and N,N-diisopropylethylamine (1.1 mL, 6.4 mmol). Theresin was then added once the reaction mixture became homogenous. Theresulting mixture was allowed to shake at 5° C. for 5 hours. The resinwas then filtered off and washed with N,N-dimethylformamide (3×20 mL)and dichloromethane (3×20 mL), and allowed to air dry to yield (042).

Compound (043):

Resin (042) (0.23 mmol) was placed in a solution of 20% piperidine inN,N-dimethylformamide (20 mL) and the resulting mixture was shaken atroom temperature for 1 hour. The resin was filtered off and washed withN,N-dimethylformamide (3×10 mL) and dichloromethane (3×10 mL).

To a 0° C. solution of (009) (203 mg, 1.6 mmol) in N,N-dimethylformamide(10 mL) were added HOBT (346 mg, 2.6 mmol), HBTU (970 mg, 2.6 mmol) andN,N-diisopropylethylamine (1.1 mL, 6.4 mmol). Once the resulting mixturebecame homogenous, the resin was added and the resulting reactionmixture was allowed to shake at room temperature overnight. The resinwas then filtered off, washed with N,N-dimethylformamide (3×20 mL) anddichloromethane (3×20 mL), and allowed to air dry to yield (043).

Compound (044):

To (043) was added a solution of 50% of trifluoroacetic acid indichloromethane (10 mL), and the resulting mixture was allowed to shakefor 30 minutes. The resin was then filtered off and washed withdichloromethane (3×10 mL). The volatiles were removed under reducedpressure to provide (044) which was characterized by LC/MS (LCRS (MH)m/z: 300.11) and used without further purification.

Compound (045):

To a 0° C. solution of aforementioned intermediates (044) and (006) (195mg, 0.64 mmol), HOBT (137 mg, 1.0 mmol) and HBTU (357 mg, 1.0 mmol) intetrahydrofuran (50 mL) was added a solution ofN,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrhydrofuran (5 mL).The mixture was stirred at room temperature for 4 hours. It was thendiluted with ethyl acetate (200 mL) and washed with saturated aqueoussodium bicarbonate (2×10 mL) and brine (10 mL). The organic layers weredried over sodium sulfate and filtered through Celite-545. The solventswere removed under reduced pressure and the residue was purified by HPLC(aqueous ammonium acetate and acetonitrile) to provide (045) (84 mg)which was characterized by LC/MS (LCRS (MH) m/z: 453.23); >80%proteasome CT-L inhibition at 20 mg/kg PO.

Example 7

Compound (047):

To a 0° C. solution of N-Boc-serine(methyl ether) (001) (6.57 g, 33mmol), L-alanine benzyl ester hydrochloride (046) (6.45 g, 30 mmol),HOBT (5.05 g, 33 mmol) and HBTU (11.8 g, 33 mmol) in tetrahydrofuran(400 mL) was added a solution of N,N-diisopropylethylamine (9.0 g, 70mmol) in tetrahydrofuran (50 mL) over 10 minutes. The mixture becamehomogenous and was stirred at room temperature for another 5 hours. Mostof the solvent was then removed under reduced pressure and the resultingmaterial diluted with ethyl acetate (500 mL). It was washed withsaturated aqueous sodium bicarbonate (2×150 mL) and brine (200 mL) andthe organic layers were dried over sodium sulfate and filtered throughCelite-545. The solvents were removed under reduced pressure and theresidue was purified by flash chromatography (hexane and ethyl acetate)to provide (047) (11.8 g) which was characterized by LC/MS (LCRS (MH)m/z: 381.19).

Compound (048):

To a 0° C. solution of (047) (11.8 g, 31.0 mmol) in dichloromethane (100mL) was added trifluoroacetic acid (50 mL) over 10 minutes, and theresulting mixture was stirred at the same temperature for another 3hours. The solvents were then removed under reduced pressure and theresidue was placed under high vacuum overnight to provide the TFA saltof (048), which was characterized by LC/MS (LCRS (MH) m/z: 281.15) andwas used without further purification.

Compound (049):

To a 0° C. solution of (048), 5-methyl-isoxazole-3-carboxylic acid (009)(3.93 g, 31 mmol), HOBT (4.7 g, 35 mmol) and HBTU (12.5 g, 35 mmol) intetrahydrofuran (400 mL) was added a solution ofN,N-diisopropylethylamine (20 mL) in tetrahydrofuran (100 mL) over 10minutes, and the pH of the resulting mixture was ˜8. The mixture wasstirred at room temperature for another 5 hours. Most of the solvent wasthen removed under reduced pressure and diluted with ethyl acetate (1.0L). It was then washed with saturated aqueous sodium bicarbonate (2×100mL) and brine (100 mL) and the organic layers were dried over sodiumsulfate and filtered through Celite-545. The solvents were removed underreduced pressure and residue was purified by flash chromatography(hexane and ethyl acetate) to provide (049) (10.8 g) which wascharacterized by LC/MS (LCRS (MH) m/z: 390.16).

Compound (044):

To a 0° C. solution of (049) (3.28 g, 8.4 mmol) in tetrahydrofuran (100mL) was added 10% Pd/C (500 mg). The resulting mixture was allowed tostir under 1 atmosphere of hydrogen for 4 hours. The mixture was thenfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The organic filtrate was concentrated under reducedpressure and placed under high vacuum for 2 hrs to yield (044), whichwas characterized by LC/MS (LCRS (MH) m/z: 281.15) and was used withoutfurther purification.

Compound (045):

To a 0° C. solution of (044) and (006) (1.9 g, 8.5 mmol), HOBT (2.0 g,13 mmol) and HBTU (5.4 g, 14 mmol) in tetrahydrofuran (200 mL) was addeda solution of N,N-diisopropylethylamine (5.4 g, 42 mmol) intetrahydrofuran (10 mL). The mixture was stirred at room temperature foranother 5 hours. Most of the solvent was then removed under reducedpressure and the resulting material diluted with ethyl acetate (400 mL).It was then washed with saturated aqueous sodium bicarbonate (2×50 mL)and brine (50 mL) and the organic layers were dried over sodium sulfateand filtered through Celite-545. The solvents were removed under reducedpressure and residue was purified by HPLC (aqueous ammonium acetate andacetonitrile) to provide (045) (1.35 g) which was characterized by LC/MS(LCRS (MH) m/z: 453.23).

Example 8

Compound (051):

To a 0° C. solution of N-Boc-L-2-pyridylalanine (050) (1.0 g, 3.76mmol), L-phenylalanine benzyl ester hydrochloride (002) (1.3 g, 3.76mmol), HOBT (0.68 g, 5.0 mmol) and HBTU (1.8 g, 5.0 mmol) intetrahydrofuran (100 mL) was added a solution ofN,N-diisopropylethylamine (1.6 mL) in tetrahydrofuran (10 mL). Themixture was stirred at room temperature for another 3 hours and thendiluted with ethyl acetate (200 mL), washed with saturated aqueoussodium bicarbonate (2×50 mL) and brine (100 mL) and the organic layerswere dried over sodium sulfate and filtered through Celite-545. Thesolvent was removed under reduced pressure and the residue was purifiedby flash chromatography (hexane and ethyl acetate) to provide (051)(1.45 g) which was characterized by LC/MS (LCRS (MH) m/z: 504.24).

Compound (052):

To a 0° C. solution of (051) in tetrahydrofuran (100 mL) was added 10%Pd/C (100 mg) and the resulting mixture was allowed to stir under 1atmosphere of hydrogen for 4 hours. The mixture was then filteredthrough Celite-545 and the filter cake was washed with tetrahydrofuran.The organic filtrate was then concentrated under reduced pressure andplaced under high vacuum to provide (052) by LC/MS (LCRS (MH) m/z:414.2) which was used without further purification.

Compound (053):

To a 0° C. solution of (052) and (006) (0.85 g, 3.9 mmol), HOBT (0.70 g,5.3 mmol) and HBTU (1.70 g, 4.9 mmol) in tetrahydrofuran (100 mL) wasadded a solution of N,N-diisopropylethylamine (3 mL) in tetrahydrofuran(10 mL) and the mixture was stirred at room temperature overnight. Itwas then diluted with ethyl acetate (200 mL) and washed with saturatedaqueous sodium bicarbonate (2×50 mL) and brine (50 mL). The organiclayers were dried over sodium sulfate and filtered through Celite-545,and the solvents were removed under reduced pressure and the residue waspurified by flash chromatography (hexane and ethyl acetate) and HPLC(aqueous ammonium acetate and acetonitrile) to provide (053) (1.51 g)which was characterized by LC/MS (LCRS (MH) m/z: 567.21).

Compound (054):

To a 0° C. solution of (053) (200 mg, 0.352 mmol) in dichloromethane (10mL) was added trifluoroacetic acid (10 mL), and the resulting solutionwas stirred at the same temperature for another hour. The solution wasconcentrated under reduced pressure and placed under high vacuum toprovide (054) as confirmed by LC/MS (LCRS (MH) m/z: 467.26) which wasused without further purification.

Compound (055):

To a 0° C. solution of (054) and 5-methyl-isoxazole-3-carboxylic acid(009) (127 mg, 1.0 mmol), HOBT (135 mg, 1.0 mmol) and HBTU (350 mg, 1.0mmol) in tetrahydrofuran (100 mL) was added a solution ofN,N-diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL). Themixture was stirred at room temperature for 5 hours. It was then dilutedwith ethyl acetate (200 mL) and washed with saturated aqueous sodiumbicarbonate (2×10 mL) and brine (10 mL). The organic layers were driedover sodium sulfate and filtered through Celite-545, the solvents wereremoved under reduced pressure and the residue was purified by HPLC(aqueous ammonium acetate and acetonitrile) to provide (055) (40 mg)which was characterized by LC/MS (LCRS (MH) m/z: 576.27); >80%proteasome CT-L inhibition at 20 mg/kg PO.

Example 9

Compound (057):

To a 0° C. solution of N-Boc-L-n-valine (056) (1.0 g, 4.6 mmol),L-phenylalanine benzyl ester hydrochloride (002) (1.4 g, 4.6 mmol), HOBT(1.0 g, 7.4 mmol) and HBTU (2.8 g, 7.4 mmol) in tetrahydrofuran (100 mL)was added a solution of N,N-diisopropylethylamine (3.2 mL, 18.4 mmol) intetrahydrofuran (10 mL). The mixture was stirred at room temperature for3 hours and then diluted with ethyl acetate (200 mL), washed withsaturated aqueous sodium bicarbonate (2×50 mL) and brine (100 mL), andthe organic layers were dried over sodium sulfate and filtered throughCelite-545. The solvent was removed under reduced pressure and theresidue was purified by flash chromatography (hexane and ethyl acetate)to provide (057) which was characterized by LC/MS (LCRS (MH) m/z:455.25).

Compound (058):

To a 0° C. solution of (057) (1.30 g, 2.875 mmol) in tetrahydrofuran(100 mL) was added 10% Pd/C (100 mg). The resulting mixture was allowedto stir under 1 atmosphere of hydrogen for 4 hours. The mixture wasfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The filtrate was then concentrated under reducedpressure and placed under high vacuum to provide (058) as confirmed byLC/MS (LCRS (MH) m/z: 365.2) which was used without furtherpurification.

Compound (059):

To a 0° C. solution of (058) and (006) (0.99 g, 4.6 mmol), HOBT (0.62 g,4.6 mmol) and HBTU (1.70 g, 4.9 mmol) in tetrahydrofuran (100 mL) wasadded a solution of N,N-diisopropylethylamine (2.4 mL) intetrahydrofuran (10 mL). The mixture was stirred at room temperatureovernight and was then diluted with ethyl acetate (200 mL) and washedwith saturated aqueous sodium bicarbonate (2×50 mL) and brine (50 mL).The organic layers were dried over sodium sulfate and filtered throughCelite-545. The solvents were then removed under reduced pressure andthe residue was purified by HPLC (aqueous ammonium acetate andacetonitrile) to provide (059) (1.21 g) which was characterized by LC/MS(LCRS (MH) m/z: 518.32).

Compound (060):

To a 0° C. solution of (059) (250 mg, 0.48 mmol) in dichloromethane (10mL) was added trifluoroacetic acid (10 mL), and the resulting solutionwas stirred at the same temperature for another hour. The organic layerswere concentrated under reduced pressure and placed under high vacuum toprovide (060) as confirmed by LC/MS (LCRS (MH) m/z: 418.26) which wasused without further purification.

Compound (061):

To a 0° C. solution of (060) and (022) (122 mg, 0.58 mmol), HOBT (104mg, 0.77 mmol) and HBTU (292 mg, 0.72 mmol) in tetrahydrofuran (100 mL)was added a solution of N,N-diisopropylethylamine (0.35 mL) intetrahydrofuran (2 mL) and the mixture was stirred at room temperaturefor another 4 hours. It was then diluted with ethyl acetate (200 mL) andwashed with saturated aqueous sodium bicarbonate (2×10 mL) and brine (10mL). The organic layers were dried over sodium sulfate and filteredthrough Celite-545. The solvents were removed under reduced pressure andthe residue was purified by HPLC (aqueous ammonium acetate andacetonitrile) to provide (061) (88.4 mg) which was characterized byLC/MS (LCRS (MH) m/z: 612.33); >80% proteasome CT-L inhibition at 40mg/kg PO.

Example 10

Compound (063):

To a 0° C. solution of N-Boc-HoSer(OMe)-OH (062) (1.0 g, 4.3 mmol),L-phenylalanine benzyl ester hydrochloride (002) (1.3 g, 4.3 mmol), HOBT(0.88 g, 6.5 mmol) and HBTU (2.3 g, 6.5 mmol) in tetrahydrofuran (100mL) was added a solution of N,N-diisopropylethylamine (2.0 mL) intetrahydrofuran (5 mL). The mixture was stirred at room temperature foranother 3 hours and then diluted with ethyl acetate (200 mL) and washedwith saturated aqueous sodium bicarbonate (2×50 mL) and brine (100 mL).The organic layers were dried over sodium sulfate and filtered throughCelite-545. The solvents were removed under reduced pressure and theresidue was purified by flash chromatography (hexane and ethyl acetate)to provide (063) (1.81 g) which was characterized by LC/MS (LCRS (MH)m/z: 471.24).

Compound (064):

To a 0° C. solution of (063) (1.35 g, 2.875 mmol) in tetrahydrofuran(100 mL) was added 10% Pd/C (100 mg). The resulting mixture was allowedto stir under 1 atmosphere of hydrogen for 4 hours. The mixture wasfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The organic filtrate was concentrated under reducedpressure and placed under high vacuum to provide (064) as confirmed byLC/MS (LCRS (MH) m/z: 381.19) which was used without furtherpurification.

Compound (065):

To a 0° C. solution of (065) and (006) (0.99 g, 4.6 mmol), HOBT (0.62 g,4.6 mmol) and HBTU (1.70 g, 4.9 mmol) in tetrahydrofuran (100 mL) wasadded a solution of N,N-diisopropylethylamine (2.4 mL) intetrahydrofuran (10 mL). The mixture was stirred at room temperatureovernight and then diluted with ethyl acetate (200 mL) and washed withsaturated aqueous sodium bicarbonate (2×50 mL) and brine (50 mL). Theorganic layers were dried over sodium sulfate and filtered throughCelite-545. The solvents were removed under reduced pressure and theresidue was purified by HPLC (aqueous ammonium acetate and acetonitrile)to provide (065) (1.11 g) which was characterized by LC/MS (LCRS (MH)m/z: 534.31).

Compound (066):

To a 0° C. solution of (065) (230 mg, 0.43 mmol) in dichloromethane (20mL) was added trifluoroacetic acid (10 mL), and the resulting solutionwas stirred at the same temperature for another one hour. The reactionmixture was then concentrated under reduced pressure and placed underhigh vacuum to provide (066) as confirmed by LC/MS (LCRS (MH) m/z:434.26) which was used without further purification.

Compound (068):

To a 0° C. solution of (066) and 5-isopropylisoxazole-3-carboxylic acid(067) (81 mg, 0.52 mmol), HOBT (93 mg, 0.69 mmol) and HBTU (262 mg, 0.69mmol) in tetrahydrofuran (100 mL) was added a solution ofN,N-diisopropylethylamine (0.30 mL) in tetrahydrofuran (2 mL) and themixture was stirred at room temperature for another 4 hours. It was thendiluted with ethyl acetate (200 mL) and washed with saturated aqueoussodium bicarbonate (2×10 mL) and brine (10 mL). The organic layers weredried over sodium sulfate and filtered through Celite-545. The solventswere removed under reduced pressure and the residue was purified by HPLC(aqueous ammonium acetate and acetonitrile) to provide (068) (75.7 mg)which was characterized by LC/MS (LCRS (MH) m/z: 571.31); >70%proteasome CT-L inhibition at 40 mg/kg PO.

Example 11

Compound (070):

To a solution of L-serine (methyl ether) hydrochloride (069) (1.0 g, 6.4mmol) in water/dioxane (1:1, 80 ml) was added sodium hydroxide (768 mg,19.2 mmol). After the mixture was stirred at room temperature for 30minutes, it was cooled to 0° C., and a solution of 9-fluorenylmethylchloroformate (1.65 g, 6.4 mmol) in dioxane (16 mL) was addeddropwisely. The reaction mixture was allowed to stir at room temperaturefor another 4 hours. The solvents were then removed, the residue wasdiluted with water and the pH was adjusted to ˜1 with 1N HCl, and theaqueous layer was extracted with ethyl acetate (4×100 mL). The organiclayers were concentrated under reduced pressure and placed under highvacuum to provide (070) (1.8 g) as confirmed by LC/MS (LCRS (MH) m/z:342.13) which was used without further purification.

Compound (071):

The resin HMPB-BHA (500 mg, 0.32 mmol) was washed with dichloromethane.In a dry flask, Fmoc-Ser(Me)-OH (070) (546 mg, 1.6 mmol) was dissolvedin dichloromethane and to the solution was added 1-methylimidazole (95μL, 1.2 mmol) followed by MSNT (474 mg, 1.6 mmol). Once the resultingmixture had become homogenous (10 minutes) it was added to the HMPB-BHAresin as a suspension in dichloromethane (5 mL). The resulting reactionmixture was allowed to shake overnight. The resin was then filtered offand washed with DMF (3×20 mL), MeOH (3×20 mL), DCM (3×20 mL), andallowed to air dry to yield (071).

Compound (072):

Resin (071) (300 mg, 0.192 mmol) was placed in a solution of 20%piperidine in N,N-dimethylformamide (20 mL) and the resulting mixturewas shaken at room temperature for 30 minutes. The resin was filteredoff and washed with N,N-dimethylformamide (3×20 mL) and dichloromethane(3×20 mL) twice.

To a 0° C. solution of Fmoc-Ser(Me)-OH (070) (0.48 mmol, 163 mg) inN,N-dimethylformamide (10 mL) was added HOBT (104 mg, 0.77 mmol), HBTU(291 mg, 0.77 mmol) and diisopropylethylamine (0.34 mL, 1.92 mmol). Oncethe resulting mixture became homogenous, the resin (0.13 mmol, 200 mg)was added and the resulting reaction mixture was allowed to shakeovernight. The resin was then filtered off and washed with DMF (10 mL),DCM (10 mL), MeOH (10 mL), H₂O (10 mL), DMF (10 mL), MeOH (10 mL), andDCM (10 mL), and allowed to air dry to yield (072).

Compound (073):

To (072) (300 mg, 0.19 mmol) was added a solution of 20% piperidine inN,N-dimethylformamide (20 mL) and the resulting mixture was shaken atroom temperature for 30 minutes. The resin was filtered off and washedwith N,N-dimethylformamide (3×20 mL) and dichloromethane (3×20 mL)twice.

To a 0° C. solution of (009) (61 mg, 0.48 mmol) in N,N-dimethylformamide(2 mL) was added HOBT (104 mg, 0.77 mmol), HBTU (291 mg 0.77 mmol) andN,N-diisopropylethylamine (0.34 mL, 1.92 mmol). Once the resultingmixture became homogenous, the resin (300 mg, 0.192 mmol) was added andthe resulting reaction mixture was allowed to shake at room temperatureovernight. The resin was then filtered off, washed with DMF (10 mL), DCM(10 mL), MeOH (10 mL), H₂O (10 mL), DMF (10 mL), MeOH (10 mL), and DCM(10 mL), and allowed to air dry to yield (073).

Compound (074):

To (073) was added a solution of 50% of trifluoroacetic acid indichloromethane (10 mL), and the resulting mixture was allowed to shakefor 30 minutes. The resin was then filtered off and washed withdichloromethane (3×10 mL). The volatiles were removed under reducedpressure, and the desired compound (074) was characterized by LC/MS(LCRS (MH) m/z: 330.12) and used without further purification.

Compound (075):

To a 0° C. solution of (074) and (006) (78 mg, 0.38 mmol), HOBT (41 mg,0.30 mmol) and HBTU (116 mg, 0.30 mmol) in acetonitrile (50 mL) wasadded a solution of N,N-diisopropylethylamine (0.1 mL, 0.6 mmol). Themixture was stirred at 0-4° C. overnight and then diluted with ethylacetate (200 mL). It was then washed with saturated aqueous sodiumbicarbonate (2×10 mL) and brine (10 mL) and the organic layers weredried over sodium sulfate and filtered through Celite-545. The solventswere removed under reduced pressure and the residue was purified by HPLC(aqueous ammonium acetate and acetonitrile) to provide (075) (29 mg)which was characterized by LC/MS (LCRS (MH) m/z: 483.24); >80%proteasome CT-L inhibition at 20 mg/kg PO.

Example 12

Compound (076):

To a 0° C. solution of N-Boc serine(methyl ether)-OH (43.8 g, 200 mmol),triethylamine (26.5 g, 260 mmol) and 4-(dimethylamino)pyridine indichloromethane (1.2 L) was added a solution of benzyl chloroformate (41g, 240 mmol) in dichloromethane (250 mL) over 30 minutes and theresulting mixture was stirred at the same temperature for another 3hours. Saturated aqueous sodium bicarbonate (200 mL) was then added andthe organic layer was washed with saturated aqueous sodium bicarbonate(200 mL) and brine (200 mL). The organic layers were dried over sodiumsulfate and filtered through Celite-545. The solvents were removed underreduced pressure and residue was purified by flash chromatography(hexane and ethyl acetate) to provide (076) (54 g) which wascharacterized by LC/MS (LCRS (MH) m/z: 310.16).

Compound (077):

To a 0° C. solution of (076) (54 g, 174.6 mmol) in dichloromethane (200mL) was added trifluoroacetic acid (200 mL) over 10 minutes, and theresulting mixture was stirred at the same temperature for another 3hours. The solvents were then removed under reduced pressure and theresidue was placed under high vacuum overnight giving the TFA salt of(077), confirmed by LC/MS (LCRS (MH) m/z: 210.11), and was used withoutfurther purification.

Compound (078):

To a 0° C. solution of (077) (43.8 g, 200 mmol), N-Boc serine(methylether)-OH (36.7 g, 167 mmol), HOBT (27 g, 200 mmol) and HBTU (71.4 g,200 mmol) in tetrahydrofuran (1.2 L) was added a solution ofN,N-diisopropylethylamine (75 g, 600 mmol) in tetrahydrofuran (250 mL)over 10 minutes, and pH of the resulting mixture was ˜8. The mixture wasstirred at room temperature for another 5 hours. Most of the solvent wasthen removed under reduced pressure and the resulting material dilutedwith ethyl acetate (1.0 L). It was then washed with saturated aqueoussodium bicarbonate (2×150 mL) and brine (200 mL) and the organic layerswere dried over sodium sulfate and filtered through Celite-545. Thesolvents were removed under reduced pressure and residue was purified byflash chromatography (hexane and ethyl acetate) to provide (078) (65 g)which was characterized by LC/MS (LCRS (MH) m/z: 411.21).

Compound (079):

To a 0° C. solution of (079) (13.4 g, 32.7 mmol) in tetrahydrofuran (300mL) was added 10% Pd/C (2.7 g) and the resulting mixture was allowed tostir under 1 atmosphere of hydrogen for 4 hours. The mixture wasfiltered through Celite-545 and the filter cake was washed withtetrahydrofuran. The organic layers were concentrated under reducedpressure and placed under high vacuum to provide (079) as confirmed byLC/MS (LCRS (MH) m/z: 321.16) which was used without furtherpurification.

Compound (080):

To a 0° C. solution of (079) and (006) (5.6 g, 26 mmol), HOBT (6.0 g,41.4 mmol) and HBTU (14.8 g, 41.4 mmol) in tetrahydrofuran (400 mL) wasadded a solution of N,N-diisopropylethylamine (23 mL) in tetrahydrofuran(40 mL) and the mixture was stirred at room temperature overnight. Mostof the solvent was then removed under reduced pressure and the resultingmaterial diluted with ethyl acetate (500 mL) and washed with saturatedaqueous sodium bicarbonate (2×100 mL) and brine (100 mL). The organiclayers were dried over sodium sulfate and filtered through Celite-545.The solvents were removed under reduced pressure and the residue waspurified by flash chromatography (hexane and ethyl acetate) to provide(080) (9.2 g) which was characterized by LC/MS (LCRS (MH) m/z: 474.27).

Compound (081):

To a 0° C. solution of (080) (200 mg, 0.43 mmol) in dichloromethane (10mL) was added trifluoroacetic acid (10 mL), and the resulting solutionwas stirred at the same temperature for another hour. The organic layerswere concentrated under reduced pressure and placed under high vacuum toprovide (081) as confirmed by LC/MS (LCRS (MH) m/z: 374.22) which wasused without further purification.

Compound (075):

To a 0° C. solution of (081) and 5-methyl-isoxazole-3-carboxylic acid(009) (65 mg, 0.5 mmol), HOBT (65 mg, 0.5 mmol) and HBTU (175 mg, 0.5mmol) in tetrahydrofuran (50 mL) was added a solution ofN,N-diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL) and themixture was stirred at room temperature for another 5 hours. It was thendiluted with ethyl acetate (200 mL) and washed with saturated aqueoussodium bicarbonate (2×10 mL) and brine (10 mL). The organic layers weredried over sodium sulfate and filtered through Celite-545. The solventswere removed under reduced pressure and the residue was purified by HPLC(aqueous ammonium acetate and acetonitrile) to provide (075) (85 mg)which was characterized by LC/MS (LCRS (MH) m/z: 483.24).

Example 13

Compound (083):

To a 0° C. solution of (081) (160 mg, 0.43 mmol) andisoxazole-3-carboxylic acid (082) (60 mg, 0.5 mmol), HOBT (65 mg, 0.5mmol) and HBTU (175 mg, 0.5 mmol) in tetrahydrofuran (50 mL) was added asolution of N,N-diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL)and the mixture was stirred at room temperature for another 5 hours. Itwas then diluted with ethyl acetate (200 mL) and washed with saturatedaqueous sodium bicarbonate (2×10 mL) and brine (10 mL). The organiclayers were dried over sodium sulfate and filtered through Celite-545.The solvents were removed under reduced pressure and the residue waspurified by HPLC (aqueous ammonium acetate and acetonitrile) to provide(083) (74 mg) which was characterized by LC/MS (LCRS (MH) m/z:469.22); >80% proteasome CT-L inhibition at 20 mg/kg PO.

Example 14

Compound (085):

To a 0° C. solution of (081) (160 mg, 0.43 mmol) andisoxazole-3-carboxylic acid (084) (65 mg, 0.5 mmol), HOBT (65 mg, 0.5mmol) and HBTU (175 mg, 0.5 mmol) in tetrahydrofuran (50 mL) was added asolution of N,N-diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL)and the mixture was stirred at room temperature for another 5 hours. Thereaction was then diluted with ethyl acetate (200 mL) and washed withsaturated aqueous sodium bicarbonate (2×10 mL) and brine (10 mL). Theorganic layers were dried over sodium sulfate and filtered throughCelite-545. The solvents were removed under reduced pressure and theresidue was purified by HPLC (aqueous ammonium acetate and acetonitrile)to provide (085) (71 mg) which was characterized by LC/MS (LCRS (MH)m/z: 483.24); >50% proteasome CT-L inhibition at 20 mg/kg PO.

Compound (087):

To a solution of (086) (prepared using the same procedure as (005)except that Cbz-phenylalanine was substituted for Cbz-Leucine) (0.100 g,0.0295 mmol) in trifluoroacetic acid (10 mL) was added 10% Pd/C (20 mg).The resulting mixture was allowed to stir under 1 atmosphere of hydrogenfor 6 hours. The mixture was then filtered through Celite-545 and thefilter cake washed with dichloromethane (50 mL). The filtrate wasconcentrated under reduced pressure and placed under high vacuumovernight to provide (087) as confirmed by LC/MS (LCRS (MH) m/z: 206.1)which was used in the subsequent transformation without furtherpurification.

Compound (088):

To a 0° C. solution of (087) and (074) (166 mg, 0.354 mmol), HOBT (54mg, 0.354 mmol) and HBTU (134 mg, 0.354 mmol) in tetrahydrofuran (20 mL)was added N,N-diisopropylethylamine (0.2 mL, 1.18 mmol). The mixture wasstirred 0° C. overnight and became homogenous. It was then diluted withethyl acetate (20 mL) and washed with saturated aqueous sodiumbicarbonate (2×10 mL) and brine (10 mL). The organic layers were driedover sodium sulfate and filtered through Celite-545 and concentratedunder reduced pressure and the residue was purified by HPLC (aqueousammonium acetate and acetonitrile) to provide (088) (10 mg) ascharacterized by LC/MS (LCRS (MH) m/z: 517.69); >80% proteasome CT-Linhibition at 20 mg/kg PO.

Compound (090):

To a solution of (089) (prepared using the same procedure as for (005)except that Cbz-4-fluorophenylalanine was substituted for Cbz-leucine)(0.100 g, 0.28 mmol) in trifluoroacetic acid (10 mL) was added 10% Pd/C(20 mg). The resulting mixture was allowed to stir under 1 atmosphere ofhydrogen for 6 hours. The mixture was filtered through Celite-545 andthe filter cake washed with dichloromethane (50 mL). The filtrate wasconcentrated under reduced pressure and placed under high vacuumovernight to provide (090) as confirmed by LC/MS (LCRS (MH) m/z: 224.1)which was used in the subsequent transformation without furtherpurification.

Compound (091):

To a 0° C. solution of (090) and (074) (110 mg, 0.336 mmol), HOBT (51mg, 0.336 mmol) and HBTU (127 mg, 0.336 mmol) in tetrahydrofuran (20 mL)was added N,N-diisopropylethylamine (0.2 mL, 1.18 mmol). The mixture wasstirred 0° C. overnight and became homogenous. It was then diluted withethyl acetate (20 mL) and washed with saturated aqueous sodiumbicarbonate (2×10 mL) and brine (10 mL). The organic layers were driedover sodium sulfate, filtered through Celite-545, and concentrated underreduced pressure. The residue was then purified by HPLC (aqueousammonium acetate and acetonitrile) to provide (091) (60 mg) which wascharacterized by LC/MS (LCRS (MH) m/z: 535.69); >80% proteasome CT-Linhibition at 20 mg/kg PO.

Biological Activity

Compounds were formulated in 10% PS80/NaCitrate (pH 3) vehicle andadministered to mice orally (PO) (3 animals/cohort). One hourpost-dosing, the animals were sacrificed and the following tissuesharvested: blood, brain, adrenal gland, heart and liver. Whole blood(˜200 μl) was washed twice with PBS and lysed by hypotonic shock (300 μl50 mM Tris pH 8, 5 mM EDTA). Blood lysates were stored at −80° C. untilassayed. Blood lysates were clarified by centrifugation in amicrocentrifuge. The CT-L specific activity of proteasome in each lysatewas evaluated by determining: a) the protein concentration by modifiedBradford assay with bovine gamma globulin as a standard; and b) the rateof cleavage of the fluorogenic proteasome substrate LLVY-AMC. Thepercent proteasome activity for the analog-treated animals wascalculated by dividing of the average specific activity for eachanalog-dosed cohort by the average specific activity of thevehicle-dosed cohort. Percent proteasome inhibition was calculated bysubtracting percent proteasome activity from 100.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

1. (canceled)
 2. A method for the treatment of cancer in a patient,comprising administering to the patient a therapeutically effectiveamount of a pharmaceutical composition comprising a compound having astructure of formula (I):

or a pharmaceutically acceptable salt thereof, wherein L is selectedfrom C═O and C═S; X is selected from O, S, NH, and N—C₁₋₆alkyl; Z isabsent, C₁₋₆ alkyl, or C₁₋₆alkoxy; R¹, R², and R³ are each independentlyselected from hydrogen, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆hydroxyalkyl, C₁₋₆ alkoxyalkyl, aryl, C₁₋₆ aralkyl, heteroaryl,heterocyclyl, C₁₋₆ heterocycloalkyl, C₁₋₆ heteroaralkyl, carbocyclyl,and C₁₋₆ carbocyclolalkyl; R⁴ is selected from hydrogen, C₁₋₆ aralkyl,and C₁₋₆ alkyl; R⁵ is heteroaryl; and R⁶ and R⁷ are independentlyselected from hydrogen, C₁₋₆ alkyl, and C₁₋₆ aralkyl and apharmaceutically acceptable diluent or carrier.
 3. The method of claim2, wherein Z is absent.
 4. The method of claim 2, wherein R⁴, R⁶, and R⁷are independently selected from hydrogen and methyl.
 5. The method ofclaim 2, wherein L is C═O.
 6. The method of claim 2, wherein R¹, R², andR³ are independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆ alkenyl,C₁₋₆ alkynyl, C₁₋₆ hydroxyalkyl, C₁₋₆ alkoxyalkyl, C₁₋₆ aralkyl, C₁₋₆heterocycloalkyl, C₁₋₆ heteroaralkyl, and C₁₋₆ carbocyclolalkyl.
 7. Themethod of claim 6, wherein any of R¹, R², and R³ are independently C₁₋₆alkyl.
 8. The method of claim 7, wherein any of R¹, R², and R³ areindependently selected from methyl, ethyl, propyl, isopropyl, butyl,sec-butyl, and isobutyl.
 9. The method of claim 6, wherein any of R¹,R², and R³ are independently propargyl.
 10. The method of claim 6,wherein any of R¹, R², and R³ are independently C₁₋₆ hydroxyalkyl. 11.The method of claim 10, wherein any of R¹, R², and R³ are independentlyselected from hydroxymethyl and hydroxyethyl.
 12. The method of claim 6,wherein any of R¹, R², and R³ are independently C₁₋₆ alkoxyalkyl. 13.The method of claim 12, wherein any of R¹, R², and R³ are independentlyselected from methoxymethyl and methoxyethyl.
 14. The method of claim 6,wherein any of R¹, R², and R³ are independently C₁₋₆ heteroaralkyl. 15.The method of claim 14, wherein any of R¹, R², and R³ are independentlyselected from imidazolylmethyl, pyrazolylmethyl, and thiazolylmethyl,and pyridylmethyl.
 16. The method of claim 6, wherein any of R¹, R², andR³ are independently cyclohexylmethyl.
 17. The method of claim 2,wherein R¹, R², and R³ are all different.
 18. The method of claim 2,wherein at least one of R¹ and R² is selected from C₁₋₆ hydroxyalkyl andC₁₋₆ alkoxyalkyl.
 19. The method of claim 18, wherein at least one of R¹and R² is C₁₋₆ alkoxyalkyl.
 20. The method of claim 19, wherein at leastone of R¹ and R² is selected from methoxymethyl and methoxyethyl. 21.The method of claim 20, wherein R³ is selected from C₁₋₆ alkyl and C₁₋₆aralkyl.
 22. The method of claim 21, wherein R³ is C₁₋₆ alkyl.
 23. Themethod of claim 22, wherein R³ is selected from methyl, ethyl,isopropyl, sec-butyl, and isobutyl.
 24. The method of claim 23, whereinR³ is isobutyl.
 25. The method of claim 24, wherein R³ is C₁₋₆ aralkyl.26. The method of claim 25, wherein R³ is phenylmethyl.
 27. The methodof claim 2, wherein R⁵ is 5- or 6-membered heteroaryl.
 28. The method ofclaim 27, wherein R⁵ is selected from isoxazole, isothiazole, furan,thiophene, oxazole, thiazole, pyrazole, or imidazole.
 29. The method ofclaim 28, wherein R⁵ is selected from isoxazole, furan, or thiophene.30. The method of claim 29, wherein R⁵ is furan or thiophene.
 31. Themethod of claim 28, wherein R⁵ is thiazole.
 32. The method of claim 2,wherein the compound is selected from:

or a pharmaceutically acceptable salt thereof.
 33. The method of claim2, wherein the pharmaceutical composition is orally bioavailable. 34.The method of claim 2, wherein the cancer is a solid tumor.
 35. Themethod of claim 2, wherein the cancer is a p53-related cancer.
 36. Amethod for treating cancer in a patient comprising administering to thepatient a pharmaceutical composition comprising a compound having thestructure:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable diluent or carrier.
 37. The method of claim 36, wherein thepharmaceutical composition is orally bioavailable.
 38. The method ofclaim 36, wherein the cancer is a solid tumor.
 39. The method of claim36, wherein the cancer is a p53-related cancer.